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Mutant and Wild Type Human α-Synucleins Assemble into Elongated Filaments with Distinct Morphologies in Vitro

1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês

10.1074/jbc.274.12.7619

ISSN

1083-351X

Autores

Benoit I. Giasson, Kunihiro Uryu, John Q. Trojanowski, Virginia M.‐Y. Lee,

Tópico(s)

Alzheimer's disease research and treatments

Resumo

α-Synuclein is a soluble presynaptic protein which is pathologically redistributed within intracellular lesions characteristic of several neurodegenerative diseases. Here we demonstrate that wild type and two mutant forms of α-synuclein linked to familial Parkinson's disease (Ala30 → Pro and Ala53 → Thr) self-aggregate and assemble into 10–19-nm-wide filaments with distinct morphologies under definedin vitro conditions. Immunogold labeling demonstrates that the central region of all these filaments are more robustly labeled than the N-terminal or C-terminal regions, suggesting that the latter regions are buried within the filaments. Since in vitrogenerated α-synuclein filaments resemble the major ultrastructural elements of authentic Lewy bodies that are hallmark lesions of Parkinson's disease, we propose that self-aggregating α-synuclein is the major subunit protein of these filamentous lesions. α-Synuclein is a soluble presynaptic protein which is pathologically redistributed within intracellular lesions characteristic of several neurodegenerative diseases. Here we demonstrate that wild type and two mutant forms of α-synuclein linked to familial Parkinson's disease (Ala30 → Pro and Ala53 → Thr) self-aggregate and assemble into 10–19-nm-wide filaments with distinct morphologies under definedin vitro conditions. Immunogold labeling demonstrates that the central region of all these filaments are more robustly labeled than the N-terminal or C-terminal regions, suggesting that the latter regions are buried within the filaments. Since in vitrogenerated α-synuclein filaments resemble the major ultrastructural elements of authentic Lewy bodies that are hallmark lesions of Parkinson's disease, we propose that self-aggregating α-synuclein is the major subunit protein of these filamentous lesions. non-amyloid component of senile plaques NAC precursor protein dementia with Lewy bodies glial cell inclusion Lewy body multisystem atrophy neuronal cytoplasmic inclusion polyacrylamide gel electrophoresis Parkinson's disease wild type 2-[N-morpholino]ethanesulfonic acid α-Synuclein is a small, 140-amino acid protein characterized by acidic stretches toward the C terminus and six repetitive, degenerate amino acid sequences of the prototype KTKEGV between amino acid residues 10 and 86 (1Clayton D.F. George J.M. Trends Neurosci. 1998; 21: 249-254Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar, 2Goedert M. Nature. 1997; 388: 232-233Crossref PubMed Scopus (93) Google Scholar). The first synuclein identified and cloned was from the electric eel Torpedo california (3Maroteaux L. Campanelli J.T. Scheller R.H. J. Neurosci. 1988; 8: 2804-2815Crossref PubMed Google Scholar). In the latter study, the authors also cloned a rat synuclein homologue that was later termed α-synuclein (4Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1994; 345: 27-32Crossref PubMed Scopus (904) Google Scholar). α-Synuclein was first associated with a human neurodegenerative disease when a 35-amino acid peptide corresponding to residues 61–95 in α-synuclein was purified from Alzheimer's disease senile plaques (5Uéda K. Fukushima H. Masliah E. Xia Y. Iwai A. Yoshimoto M. Otero D.A. Kondo J. Ihara Y. Saitoh T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11282-11286Crossref PubMed Scopus (1228) Google Scholar). This peptide was referred to as the non-amyloid component of senile plaques (NAC)1 and its precursor protein, i.e. α-synuclein, was designated NACP (5Uéda K. Fukushima H. Masliah E. Xia Y. Iwai A. Yoshimoto M. Otero D.A. Kondo J. Ihara Y. Saitoh T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11282-11286Crossref PubMed Scopus (1228) Google Scholar). The zebra finch homologue of α-synuclein, synelfin, was identified as a gene product potentially implicated in neuronal plasticity (6George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (730) Google Scholar). α-Synuclein is predominantly a neuronal protein expressed in brain and localized in axon terminals (5Uéda K. Fukushima H. Masliah E. Xia Y. Iwai A. Yoshimoto M. Otero D.A. Kondo J. Ihara Y. Saitoh T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11282-11286Crossref PubMed Scopus (1228) Google Scholar, 6George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (730) Google Scholar, 7Iwai A. Masliah E. Yoshimoto M. Ge N. Flanagan L. Rohan de Silva H.A. Kittel A. Saitoh T. Neuron. 1995; 14: 467-475Abstract Full Text PDF PubMed Scopus (1127) Google Scholar, 8Hong L. Ko H.W. Gwag B.J. Joe E. Lee S. Kim Y.-T. Suh Y.-H. Neuroreport. 1998; 9: 1239-1243Crossref PubMed Scopus (34) Google Scholar). Because α-synuclein is in close proximity to and is loosely associated with presynaptic vesicles, it may modulate the function of synaptic vesicles (6George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (730) Google Scholar, 7Iwai A. Masliah E. Yoshimoto M. Ge N. Flanagan L. Rohan de Silva H.A. Kittel A. Saitoh T. Neuron. 1995; 14: 467-475Abstract Full Text PDF PubMed Scopus (1127) Google Scholar). Although α-synuclein has very little structure in aqueous solution (9Weinreb P.H. Zhen W.G. Poon A.W. Conway K.A. Lansbury P.T. Biochemistry. 1996; 35: 13709-13715Crossref PubMed Scopus (1321) Google Scholar), it associates with small unilamellar acidic phospholipid vesicles in vitro and acquires increased α-helicity (10Davidson W.S. Jonas A. Clayton D.F. Georges J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1251) Google Scholar). Thus, it has been proposed that α-synuclein may bind vesicles by forming amphipathic helices similar to apolipoproteins (6George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (730) Google Scholar). A link between α-synuclein and Parkinson's disease (PD) first surfaced when Polymeropoulos et al. (11Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. Lazzarini A.M. Duvoisin R.C. Di Lorio G. Golbe L.I. Nussbau R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6667) Google Scholar) reported a missense mutation (Ala53 → Thr) in α-synuclein in four Italian and Greek kindreds with autosomal dominant PD. This finding was followed by the demonstration of α-synuclein immunoreactivity in Lewy bodies (LBs) and Lewy neurites in patients with sporadic PD and dementia with LBs (DLB) (12Spillantini M.G. Schmidt M.L. Lee V.M.-Y. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6178) Google Scholar). Significantly, these lesions are pathological hallmarks of PD and DLB (13Forno L.S. J. Neuropathol. Exp. Neurol. 1996; 55: 259-272Crossref PubMed Scopus (1244) Google Scholar, 14McKeith I.G. Galasko D. Kosaka K. Perry E.K. Dickson D.W. Hansen L.A. Salmon D.P. Lowe J. Mirra S.S. Byrne E.J. Lennox G. Quinn N.P. Edwardson J.A. Ince P.G. Bergeron C. Burns A. Miller B.L. Lovestone S. Collerton D. Jansen E.N.H. Ballard C. deVos R.A.I. Wilcock G.K. Jellinger K.A. Perry R.H. Neurology. 1996; 47: 1113-1124Crossref PubMed Scopus (3632) Google Scholar). Subsequent studies confirmed that normal, truncated, and aggregated α-synuclein are major components of LBs and Lewy neurites and that antibodies to α-synuclein are the most reliable and consistent immunological probes for detecting these lesions in situ (15Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M.-Y. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar, 16Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2420) Google Scholar, 17Takeda A. Mallory M. Sundsmo M. Honer W. Hansen L. Masliah E. Am. J. Pathol. 1998; 152: 367-372PubMed Google Scholar, 18Wakabayashi K. Matsumoto K. Takayama K. Yoshimoto M. Takahashi H. Neurosci. Lett. 1997; 239: 45-48Crossref PubMed Scopus (286) Google Scholar, 19Izizarry M.C. Growdon W. Gomez-Isla T. Newell K. George J.M. Clayton D.F. Hyman B.T. J. Neuropathol. Exp. Neurol. 1998; 57: 334-337Crossref PubMed Scopus (360) Google Scholar). More recently, α-synuclein was also shown to be a prominent component of glial cell inclusions (GCIs) and neuronal cytoplasmic inclusions (NCIs) that are characteristic of multisystem atrophy (MSA) and Hallervorden-Spatz disease (20Tu P.H. Galvin J.E. Baba M. Giasson B. Tomita T. Leight S. Nakajo S. Iwatsubo T. Trojanowski J.Q. Lee V.M.-Y. Ann. Neurol. 1998; 44: 415-422Crossref PubMed Scopus (586) Google Scholar, 21Spillantini M.G. Crowther R.A. Jakes R. Cairns N.J. Lantos P.L. Goedert M. Neurosci. Lett. 1998; 251: 205-208Crossref PubMed Scopus (827) Google Scholar, 22Wakabayashi K. Yoshimoto M. Tsuji S. Takahashi H. Neurosci. Lett. 1998; 249: 180-182Crossref PubMed Scopus (527) Google Scholar, 23Arima K. Uéda K. Sunohara N. Arakawa K. Hirai S. Nakamura M. Tonozuka-Uehara H. Kawai M. Acta Neuropathol. 1998; 96: 439-444Crossref PubMed Scopus (233) Google Scholar, 24Wakabayashi K. Hayashi S. Kakita A. Yamada M. Toyoshima Y. Yoshimoto M. Takahashi H. Acta Neuropathol. 1998; 96: 445-452Crossref PubMed Scopus (317) Google Scholar). Furthermore, a second pathogenic mutation in α-synuclein (Ala30 → Pro) also was reported in another familial PD kindred (25Krüger R. Kuhn W. Müller T. Woitalla D. Graeber M. Kösel S. Przuntek H. Epplen J.T. Schöls L. Riess O. Nat. Gen. 1998; 18: 106-107Crossref PubMed Scopus (3321) Google Scholar). Since it is unclear how α-synuclein, a very soluble protein, ends up in cellular inclusions, we carried out studies to determine whether wild type (WT) and/or mutants of α-synuclein can self-aggregatein vitro. Here, we report that the SDS solubility of wild type and mutant α-synucleins is reduced after incubation in aqueous solution at physiological temperature and this change in physical property was attributed to the formation of elongated filaments. Human WT, A30P, and A53T α-synuclein cDNAs subcloned into the bacterial expression vector pRK172 were expressed in Escherichia coliBL21 (DE3). Bacterial pellets were resuspended in high-salt lysis buffer (0.75 m NaCl, 100 mm MES, pH 7.0, 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 dialyzed against 10 mm Tris, pH 7.5, applied to a Mono Q column, 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. Proteins were resolved on slab gels by SDS-polyacrylamide gel electrophoresis (PAGE) (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar) and electrophoretically transferred onto nitrocellulose membranes (Schleicher and Schuell) in buffer containing 48 mm Tris, 39 mm glycine, and 10% methanol. Membranes were blocked with a 1% solution of powdered skim milk dissolved in Tris buffered saline-Tween (50 mm Tris, pH 7.6, 150 mm NaCl and 0.1% Tween 20), incubated with anti-α-synuclein antibody LB509 (15Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M.-Y. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar), followed with a goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Jackson ImmunoResearch Laboratories, Inc.) and developed with 3,3′-diaminobenzidine. Following incubation at 37 °C in 100 mm sodium acetate, pH 7.0, with continuous shaking, samples were centrifuged at 150,000 × g for 30 min, SDS-sample buffer (10 mm Tris, pH 6.8, 1 mmEDTA, 40 mm dithiothreitol, 1% SDS, 10% sucrose) was added to pellets and supernatants and heated to 100 °C for 15 min. α-Synuclein was resolved on SDS-PAGE, stained with Coomassie Brilliant Blue R-250, and quantified by densitometry. SNL-1 and SNL-4 are affinity-purified rabbit polyclonal antibodies raised against peptides corresponding to amino acid residues 104–119 and 2–12 in α-synuclein, respectively. Syn 202, Syn 204, Syn 205 and Syn 208 are novel mouse monoclonal antibodies raised to synucleins, as described previously (20Tu P.H. Galvin J.E. Baba M. Giasson B. Tomita T. Leight S. Nakajo S. Iwatsubo T. Trojanowski J.Q. Lee V.M.-Y. Ann. Neurol. 1998; 44: 415-422Crossref PubMed Scopus (586) Google Scholar). The epitopes for Syn 202 and 205 are localized to amino acid residues 130–140, while the Syn 204 and 208 epitopes map to residues 87–110. The relative activities of the antibodies were determined by enzyme-linked immunosorbent assay using α-synuclein as the antigen and by performing serial dilutions of the antibodies. α-Synuclein filaments were decorated with anti-α-synuclein antibodies and negative stained with uranyl acetate as described previously (27Balin B.J. Clark E.A. Trojanowski J.Q. Lee V.M.-Y. Brain Res. 1991; 556: 181-195Crossref PubMed Scopus (34) Google Scholar). Briefly, assembled α-synuclein filaments were absorbed to 300 mesh carbon coated copper grids and stained with 1% uranyl acetate or labeled with antibodies to α-synuclein followed by secondary antibodies conjugated to 10 nm gold and staining with 1% uranyl acetate. The purity of recombinant α-synuclein proteins was demonstrated in Coomassie Blue-stained SDS-PAGE gels (Fig.1 A). No contaminating proteins were seen even when 100 μg of purified recombinant α-synucleins were loaded in separate lanes of an SDS-PAGE gel (data not shown). Incubation of 5 mg/ml WT α-synuclein at 37 °C for 48 h in a number of different buffers resulted in the aggregation of α-synuclein as reflected by decreased mobility on SDS-PAGE (Fig.1 B). Diffuse smears of the incubated proteins on SDS-PAGE gels may reflect the rapid association/dissociation of α-synuclein in the presence of SDS. Temperature was a major determinant of α-synuclein aggregation, since under the same conditions, incubation at 37 °C generated abundant aggregation that was not significantly detected when the protein was incubated at room temperature (Fig.1 B). WT (Fig. 1 C), A30P (Fig. 1 D), and A53T (Fig. 1 E) α-synucleins demonstrated a similar ability to aggregate, although A53T α-synuclein seemed to aggregate to a slightly greater extent at lower concentrations. The ability of α-synuclein to polymerize was confirmed with centrifugal sedimentation experiments (Fig. 2). WT, A30P, and A53T α-synuclein polymerization was concentration- and time-dependent, and the A53T mutant had a greater propensity to polymerize.Figure 2α-Synuclein polymerization is time- and concentration-dependent. WT, A30P, and A53T α-synucleins were incubated at 37 °C in 100 mm sodium acetate, pH 7.0, for 0–48 h. The proteins were sedimented as described under "Experimental Procedures," and the percentage of each protein in the pellet is indicated on the y axis. The protein concentrations used for each incubation are indicated at thebottom of each bar graph; n = 2.The range for each set of experiments was less than ±13% from the mean.View Large Image Figure ViewerDownload (PPT) Electron microscopic analysis of wild type and mutant α-synucleins postincubation revealed that they formed elongated filaments that frequently attained lengths of several microns (Fig.3). In some fields, a plethora of α-synuclein filaments filled the whole area on the grid (Fig.3 A). Interestingly, the morphology of the different synuclein filaments varied. For example, WT α-synuclein mainly formed straight filaments, although twisted filaments were also observed (Fig.3 B). In contrast, A53T α-synuclein predominantly formed twisted filaments that appeared to contain two protofilaments in a regular helical fibril (Fig. 3 C), while A30P α-synuclein formed filaments that were straight (Fig. 3 D). WT and A30P α-synuclein filaments had diameters ranging between 10 and 15 nm (mean = 12 ± 1.4 nm) and 11–16 nm (mean = 13 ± 1.4 nm), respectively, whereas A53T α-synuclein filaments were slightly wider with widths ranging between 16 and 19 nm (mean = 17 ± 1.1 nm). α-Synuclein filaments were only modestly labeled with antibodies to the N terminus (SNL-4) (Fig.4 A) or the C-terminal region (SNL-1; Syn 202 and Syn 205) (Fig. 4, B and C; data not shown) of α-synuclein. However, antibodies to epitopes within the central part of α-synuclein (Syn 204 and Syn 208) demonstrated very strong labeling (Fig. 4, D–F). Antibodies SNL-1, SNL-4, Syn 202, and Syn 205 were used at 10–20-fold higher relative immunoreactiveactivity than antibodies Syn 204 and Syn 208. Thus, these results may suggest that the N- and C-terminal regions of α-synuclein are less accessible than the central region within these filaments, and this may imply that both ends of the polypeptide are involved in polymer formation and embedded within the filaments. Despite the fact that α-synuclein is a small soluble synaptic protein that is largely devoid of secondary structure in aqueous buffer, we show here that mutant and WT recombinant α-synucleins polymerize into morphologically distinct filaments under a variety ofin vitro conditions. Furthermore, α-synuclein polymerization is temperature-, concentration-, and time-dependent, and the protein subunits are topographically organized within these polymers as reflected by the paucity of immunoreactivity for antibodies to α-synuclein epitopes at both ends of the polypeptide relative to those in the central region. Although a recent report demonstrated that wild-type α-synuclein forms Thioflavin-S-reactive aggregates and filaments, especially at elevated temperature (28Hashimoto M. Hsu L.J. Sisk A. Xia Y. Takeda A. Sundsmo M. Masliah E. Brain Res. 1998; 799: 301-306Crossref PubMed Scopus (249) Google Scholar), our studies substantially extend these preliminary observations by comparing the morphology of filaments formed from WT and mutant α-synucleins as well as the topography of α-synuclein in these filaments. A possible model consistent with these observations is that α-synuclein exists in numerous conformational states in aqueous solution, but when molecules with conformations compatible with dimerization interact, they may stabilize each other in this dimerized conformation, and these dimers may then serve as seeds for polymerization. The findings presented here and in previous reports suggest that α-synuclein is the major building block of the filaments that form LBs. First, anti-α-synuclein antibodies stain LBs more intensely and consistently than any other antibodies, including anti-ubiquitin antibodies (12Spillantini M.G. Schmidt M.L. Lee V.M.-Y. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6178) Google Scholar, 15Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M.-Y. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar, 16Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2420) Google Scholar, 17Takeda A. Mallory M. Sundsmo M. Honer W. Hansen L. Masliah E. Am. J. Pathol. 1998; 152: 367-372PubMed Google Scholar, 19Izizarry M.C. Growdon W. Gomez-Isla T. Newell K. George J.M. Clayton D.F. Hyman B.T. J. Neuropathol. Exp. Neurol. 1998; 57: 334-337Crossref PubMed Scopus (360) Google Scholar). Second, anti-α-synuclein immunoreactivity also is abundant in pale bodies (19Izizarry M.C. Growdon W. Gomez-Isla T. Newell K. George J.M. Clayton D.F. Hyman B.T. J. Neuropathol. Exp. Neurol. 1998; 57: 334-337Crossref PubMed Scopus (360) Google Scholar, 24Wakabayashi K. Hayashi S. Kakita A. Yamada M. Toyoshima Y. Yoshimoto M. Takahashi H. Acta Neuropathol. 1998; 96: 445-452Crossref PubMed Scopus (317) Google Scholar), which are believed to be precursor lesions of LBs. Third, neurofilaments, another filamentous component of LBs (29Goldman J.E. Yen S.H. Chiu F.C. Peress N.S. Science. 1983; 221: 1082-1084Crossref PubMed Scopus (215) Google Scholar, 30Schmidt M.L. Murray J. Lee V.M.-Y. Hill W.D. Wertkin A. Trojanowski J.Q. Am. J. Pathol. 1991; 139: 53-65PubMed Google Scholar, 31Hill W.D. Lee V.M.-Y. Hurtig H.I. Murray J.M. Trojanowski J.Q. J. Comp. Neurol. 1991; 309: 150-160Crossref PubMed Scopus (104) Google Scholar), are detected only in a small percentage of pale bodies (32Dale G.E. Probst A. Luthert P. Martin J. Anderton B.H. Leigh P.N. Acta Neuropathol. 1992; 83: 525-529Crossref PubMed Scopus (88) Google Scholar), and most anti-neurofilament antibodies only label a subset of LBs (30Schmidt M.L. Murray J. Lee V.M.-Y. Hill W.D. Wertkin A. Trojanowski J.Q. Am. J. Pathol. 1991; 139: 53-65PubMed Google Scholar). However, at least one anti-neurofilament antibody that is specific for the mid-size neurofilament subunit (RMO32) labels nearly all cortical LBs (30Schmidt M.L. Murray J. Lee V.M.-Y. Hill W.D. Wertkin A. Trojanowski J.Q. Am. J. Pathol. 1991; 139: 53-65PubMed Google Scholar). Fourth, the filaments generated in vitro from WT and mutant α-synucleins are morphologically similar to the LB filaments visualized in situ (30Schmidt M.L. Murray J. Lee V.M.-Y. Hill W.D. Wertkin A. Trojanowski J.Q. Am. J. Pathol. 1991; 139: 53-65PubMed Google Scholar, 31Hill W.D. Lee V.M.-Y. Hurtig H.I. Murray J.M. Trojanowski J.Q. J. Comp. Neurol. 1991; 309: 150-160Crossref PubMed Scopus (104) Google Scholar, 33Duffy P.E. Tennyson V.M. J. Neuropathol. Exp. Neurol. 1965; 24: 398-414Crossref Scopus (269) Google Scholar) as well as to synuclein filaments recovered from Sarkosyl-insoluble preparations of DLB brains (16Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2420) Google Scholar), while normal neurofilaments have side arms (27Balin B.J. Clark E.A. Trojanowski J.Q. Lee V.M.-Y. Brain Res. 1991; 556: 181-195Crossref PubMed Scopus (34) Google Scholar, 34Hirokawa N. Glicksman M.A. Willard M.B. J. Cell Biol. 1984; 98: 1523-1536Crossref PubMed Scopus (391) Google Scholar) that are not seen in LB fibrils (31Hill W.D. Lee V.M.-Y. Hurtig H.I. Murray J.M. Trojanowski J.Q. J. Comp. Neurol. 1991; 309: 150-160Crossref PubMed Scopus (104) Google Scholar, 33Duffy P.E. Tennyson V.M. J. Neuropathol. Exp. Neurol. 1965; 24: 398-414Crossref Scopus (269) Google Scholar). LB filament formation in vivo may result from α-synuclein accumulation, and this may be due to a reduction in the fast axonal transport (35Jensen P.H. Nielson M.S. Jakes R. Dotti C.G. Goedert M. J. Biol. Chem. 1998; 273: 26292-26294Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar) or overexpression of α-synuclein. Furthermore, once α-synuclein reaches a critical intracellular concentration, it may polymerize into filaments that aggregate into LBs or LB precursors which entrap other cytoplasmic components, and similar mechanisms may lead to the formation of Lewy neurites. Additionally, the reduced ability of A30P α-synuclein to bind vesicles (35Jensen P.H. Nielson M.S. Jakes R. Dotti C.G. Goedert M. J. Biol. Chem. 1998; 273: 26292-26294Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar) may play a mechanistic role in the onset/progression of some forms of familial PD, while the A53T mutation may be pathogenic because it increases the propensity of α-synuclein to polymerize into LB filaments (see Figs.1, C–E, and 2). The accelerated formation of filaments as a consequence of the A53T mutation has also been observed in a recent publication by Conway et al. (42Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1263) Google Scholar). Interestingly, Crowtheret al. (43Crowther R.A. Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1998; 436: 309-312Crossref PubMed Scopus (353) Google Scholar) have reported that carboxyl-terminally truncated α-synuclein may be more prone to form filaments than the full-length protein. Although the pathological implications of the latter finding is still unclear, it is possible that aberrant proteolysis of α-synuclein may create a pool of these shorter products that may play a role in initiating α-synuclein filament assembly. Although α-synuclein has been reported to be expressed predominantly in neurons, it also is a major component of GCIs and NCIs in MSA brains (20Tu P.H. Galvin J.E. Baba M. Giasson B. Tomita T. Leight S. Nakajo S. Iwatsubo T. Trojanowski J.Q. Lee V.M.-Y. Ann. Neurol. 1998; 44: 415-422Crossref PubMed Scopus (586) Google Scholar, 21Spillantini M.G. Crowther R.A. Jakes R. Cairns N.J. Lantos P.L. Goedert M. Neurosci. Lett. 1998; 251: 205-208Crossref PubMed Scopus (827) Google Scholar, 22Wakabayashi K. Yoshimoto M. Tsuji S. Takahashi H. Neurosci. Lett. 1998; 249: 180-182Crossref PubMed Scopus (527) Google Scholar, 23Arima K. Uéda K. Sunohara N. Arakawa K. Hirai S. Nakamura M. Tonozuka-Uehara H. Kawai M. Acta Neuropathol. 1998; 96: 439-444Crossref PubMed Scopus (233) Google Scholar, 24Wakabayashi K. Hayashi S. Kakita A. Yamada M. Toyoshima Y. Yoshimoto M. Takahashi H. Acta Neuropathol. 1998; 96: 445-452Crossref PubMed Scopus (317) Google Scholar). Ultrastructurally, GCIs and NCIs have been reported to be composed of 15–40-nm filaments (23Arima K. Uéda K. Sunohara N. Arakawa K. Hirai S. Nakamura M. Tonozuka-Uehara H. Kawai M. Acta Neuropathol. 1998; 96: 439-444Crossref PubMed Scopus (233) Google Scholar, 36Abe H. Yagishita S. Amano N. Iwabuchi K. Hasegawa K. Kowa K. Acta Neuropathol. 1992; 84: 273-277Crossref PubMed Scopus (70) Google Scholar, 37Murayama S. Arima K. Nakazato Y. Satoh J. Oda M. Inose T. Acta Neuropathol. 1992; 84: 32-38Crossref PubMed Scopus (80) Google Scholar, 38Kato S. Nakamura H. Hirano A. Ito H. Llena J.F. Yen S.-H. Acta Neuropathol. 1991; 82: 488-493Crossref PubMed Scopus (79) Google Scholar, 39Kato S. Nakamura H. Acta Neuropathol. 1990; 79: 584-594Crossref PubMed Scopus (129) Google Scholar, 40Arima A. Murayama S. Mukoyama M. Inose T. Acta Neuropathol. 1992; 83: 453-460Crossref PubMed Scopus (94) Google Scholar, 41Papp M.I. Kahn J.E. Lantos P.L. J. Neurol. Sci. 1989; 94: 79-100Abstract Full Text PDF PubMed Scopus (836) Google Scholar), but some GCIs appear to contain tubular filaments (38Kato S. Nakamura H. Hirano A. Ito H. Llena J.F. Yen S.-H. Acta Neuropathol. 1991; 82: 488-493Crossref PubMed Scopus (79) Google Scholar, 41Papp M.I. Kahn J.E. Lantos P.L. J. Neurol. Sci. 1989; 94: 79-100Abstract Full Text PDF PubMed Scopus (836) Google Scholar), although this issue remains controversial (36Abe H. Yagishita S. Amano N. Iwabuchi K. Hasegawa K. Kowa K. Acta Neuropathol. 1992; 84: 273-277Crossref PubMed Scopus (70) Google Scholar). Recently, the filaments in GCIs have been shown to be intensely labeled with anti-α-synuclein antibodies at the ultrastructural level (15Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M.-Y. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar, 23Arima K. Uéda K. Sunohara N. Arakawa K. Hirai S. Nakamura M. Tonozuka-Uehara H. Kawai M. Acta Neuropathol. 1998; 96: 439-444Crossref PubMed Scopus (233) Google Scholar), and Sarkosyl-insoluble α-synuclein filaments with straight and twisted morphologies similar to our synthetic filaments also have been observed in extracts of MSA brains (21Spillantini M.G. Crowther R.A. Jakes R. Cairns N.J. Lantos P.L. Goedert M. Neurosci. Lett. 1998; 251: 205-208Crossref PubMed Scopus (827) Google Scholar). Perhaps the tubular appearance of some GCI fibrils is due to the incorporation of other proteins into α-synuclein filaments or these tubular fibrils may reflect the presence of microtubules trapped within bundles of α-synuclein filaments. This uncertainty notwithstanding, we speculate that the anomalous expression, accumulation, and subsequent polymerization of α-synuclein lead to the formation of GCIs in MSA brain. However, future research will be needed to determine whether α-synuclein polymers serve as a proteinaceous trap and if α-synuclein co-assembles with other proteins previously co-localized in LBs and GCIs in situ. Finally, efforts to over express WT and mutant α-synucleins in vivo will help to elucidate the role of α-synuclein filaments in the pathogenesis of PD, DLB, MSA, and related α-synucleinopathies. We thank Dr. M. Goedert for the α-synuclein cDNA constructs and epitope localization; Drs. B. Balin, D. Murphy, and P. Sterling for help with electron microscopy; and Dr. S. Rueter for critical reading of this manuscript.

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