Portal de Periódicos da CAPES

α-Synuclein Fibrillogenesis Is Nucleation-dependent

1999; Elsevier BV; Volume: 274; Issue: 28 Linguagem: Inglês

10.1074/jbc.274.28.19509

1083-351X

Stephen Wood, Jette Wypych, Shirley Steavenson, Jean‐Claude Louis, Martin Citron, Anja Leona Biere,

Neurological diseases and metabolism

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major components of which are filaments consisting of α-synuclein. Two recently identified point mutations in α-synuclein are the only known genetic causes of PD. α-Synuclein fibrils similar to the Lewy body filaments can be formedin vitro, and we have shown recently that both PD-linked mutations accelerate their formation. This study addresses the mechanism of α-synuclein aggregation: we show that (i) it is a nucleation-dependent process that can be seeded by aggregated α-synuclein functioning as nuclei, (ii) this fibril growth follows first-order kinetics with respect to α-synuclein concentration, and (iii) mutant α-synuclein can seed the aggregation of wild type α-synuclein, which leads us to predict that the Lewy bodies of familial PD patients with α-synuclein mutations will contain both, the mutant and the wild type protein. Finally (iv), we show that wild type and mutant forms of α-synuclein do not differ in their critical concentrations. These results suggest that differences in aggregation kinetics of α-synucleins cannot be explained by differences in solubility but are due to different nucleation rates. Consequently, α-synuclein nucleation may be the rate-limiting step for the formation of Lewy body α-synuclein fibrils in Parkinson's disease. Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major components of which are filaments consisting of α-synuclein. Two recently identified point mutations in α-synuclein are the only known genetic causes of PD. α-Synuclein fibrils similar to the Lewy body filaments can be formedin vitro, and we have shown recently that both PD-linked mutations accelerate their formation. This study addresses the mechanism of α-synuclein aggregation: we show that (i) it is a nucleation-dependent process that can be seeded by aggregated α-synuclein functioning as nuclei, (ii) this fibril growth follows first-order kinetics with respect to α-synuclein concentration, and (iii) mutant α-synuclein can seed the aggregation of wild type α-synuclein, which leads us to predict that the Lewy bodies of familial PD patients with α-synuclein mutations will contain both, the mutant and the wild type protein. Finally (iv), we show that wild type and mutant forms of α-synuclein do not differ in their critical concentrations. These results suggest that differences in aggregation kinetics of α-synucleins cannot be explained by differences in solubility but are due to different nucleation rates. Consequently, α-synuclein nucleation may be the rate-limiting step for the formation of Lewy body α-synuclein fibrils in Parkinson's disease. Parkinson's disease wild type Parkinson's disease (PD)1 is a neurodegenerative disorder that predominantly affects dopaminergic neurons in the nigrostriatal system but also several other regions of the brain. A pathological hallmark of PD are Lewy bodies (1Lewy F.H. Lewandowski M. Handbuch der Neurologie. Springer, Berlin1912: 920-933Google Scholar, 2Pollanen M.S. Dickson D.W. Bergeron C. J. Neuropathol. Exp. Neurol. 1993; 52: 183-191Crossref PubMed Scopus (390) Google Scholar, 3Forno L.S. J. Neuropathol. Exp. Neurol. 1996; 55: 259-272Crossref PubMed Scopus (1256) Google Scholar), which also accumulate in dementia with Lewy bodies (4Spillantini G.M. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. 1998; 95: 6469-6473Crossref PubMed Scopus (2443) Google Scholar) and multiple system atrophy (5Arima K. Uéda K. Sunohara N. Arakawa K. Hirai S. Nakamura M. Tonozuka-Uehara H. Kawai M. Acta Neuropathol. 1998; 96: 439-444Crossref PubMed Scopus (235) Google Scholar, 6Wakabayashi K. Hayashi S. Kakita A. Yamada M. Toyoshima Y. Yoshimoto M. Takahashi H. Acta Neuropathol. 1998; 96: 445-452Crossref PubMed Scopus (320) Google Scholar), but not in a variety of other neurodegenerative disorders. The major filamentous component of Lewy bodies is α-synuclein (4Spillantini G.M. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. 1998; 95: 6469-6473Crossref PubMed Scopus (2443) Google Scholar, 7Arai T. Uéda K. Ikeda K. Akiyama H. Haga C. Kondo H. Kuroki N. Niizato K. Iritani S. Tsuchiya K. Neurosci. Lett. 1999; 259: 83-86Crossref PubMed Scopus (76) Google Scholar), a 140-amino acid protein (8Uéda K. Fukushima H. Masliah E. Xia Y. Iwai A. Yoshimoto M. Otero D. Kondo J. Ihara Y. Saitoh T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11282-11286Crossref PubMed Scopus (1239) Google Scholar). Lately, two dominant mutations in α-synuclein causing familial early onset PD have been described (9Polymeropoulos 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. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6734) Google Scholar,10Krüger R. Kuhn W. Müller T. Woitalla D. Graeber M. Kösel S. Przuntek H. Epplen J.T. Schöls L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3344) Google Scholar), suggesting that Lewy bodies contribute mechanistically to the degeneration of neurons in PD. Very recent in vitro studies have shown that recombinant α-synuclein can indeed form Lewy body-like fibrils (11Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1271) Google Scholar, 12El-Agnaf O. Jakes R. Curran M. Wallace A. FEBS Lett. 1998; 440: 67-70Crossref PubMed Scopus (241) Google Scholar, 13Hashimoto M. Hsu L. Sisk A. Xia Y. Takeda A. Sundsmo M. Masliah E. Brain Res. 1998; 799: 301-306Crossref PubMed Scopus (249) Google Scholar, 14Giasson B.I. Uryu K. Trojanowski J.Q. Lee V.M.-Y. J. Biol. Chem. 1999; 274: 7619-7622Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 15Narhi 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. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Most importantly, both PD-linked α-synuclein mutations accelerate this aggregation process (11Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1271) Google Scholar, 15Narhi 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. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar), which immediately suggests that such in vitro studies may have relevance for PD pathogenesis. We therefore decided to address the kinetic mechanism of α-synuclein fibrillogenesis. We have shown before that in a complete aggregation time course α-synuclein aggregation is slow and displays a distinct lag phase (15Narhi 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. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). This might be indicative of a nucleation-dependent polymerization mechanism consisting of an initial lag phase (nucleation) followed by a growth phase (elongation) and a steady state phase in which the ordered aggregate and monomer are at equilibrium. In the lag phase a supersaturated protein solution remains stable while soluble pre-nucleus oligomers build up. Once nuclei are formed, the aggregates grow rapidly (elongation phase) until a thermodynamic equilibrium between aggregate and monomer is reached. Under these steady state conditions the growth equilibrium constant describes the solubility of the protein, which is equivalent to its critical concentration (16Andreu J.M. Timasheff S.N. Methods Enzymol. 1986; 130: 47-59Crossref PubMed Scopus (79) Google Scholar). At concentrations above the critical concentration the nucleation step can be bypassed by the addition of exogenous nuclei (17, 18, 20, for review see Ref. 19Harper J.D. Lansbury P.T. Annu. Rev. Biochem. 1997; 66: 385-407Crossref PubMed Scopus (1420) Google Scholar). To rigorously demonstrate the nucleation dependence of α-synuclein aggregation, we needed to show a lag phase, a seeding effect, and a critical concentration of monomer at equilibrium. We report here that α-synuclein aggregation fulfills all criteria of a nucleation-dependent polymerization process. In this regard α-synuclein fibril formation resembles that of β-amyloid (Aβ) fibers (20Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1768) Google Scholar, 21Lomakin A. Chung D.S. Benedek G.B. Kirschner D.A. Teplow D.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1125-1129Crossref PubMed Scopus (736) Google Scholar, 22Wood S.J. Chan W. Wetzel R. Biochemistry. 1996; 35: 12623-12628Crossref PubMed Scopus (106) Google Scholar) and paired helical filaments (18Friedhoff P. von Bergen M. Mandelkow E.-M. Davies P. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15712-15717Crossref PubMed Scopus (287) Google Scholar), two protein aggregates characteristic for Alzheimer's disease. Interestingly, the critical concentrations of wild type and mutant α-synuclein do not differ significantly, suggesting that the accelerated aggregation of the α-synuclein mutations is not due to a decreased solubility of mutant monomer or increased stability of mutant fiber, respectively, but rather due to different nucleation rates. Therefore, α-synuclein nucleation may be the rate-limiting step for the formation of Lewy body α-synuclein fibrils in Parkinson's disease. In this light it was of interest to show that nuclei formed of mutant α-synuclein can function as a seed for elongation by wild type α-synuclein, which is a situation similar to the one found in familial PD cases. Bacterial expression and purification of α-synuclein was done as described before (15Narhi 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. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Briefly, Escherichia coli cell paste was homogenized in 20 mm Tris, 100 mm NaCl, pH 7.5, with protease inhibitor mixture Complete (Roche Molecular Biochemicals). Cells in suspension were broken by passaging through a Microfluidizer, and a clarified lysate supernatant was collected after centrifugation at 18,000 × g for 45 min. E. colicontaminating proteins were precipitated by acid precipitation of the lysate supernatant. The pH was adjusted to 3.5, and after stirring for 20–30 min, the mixture was centrifuged for 1 h at 27,000 ×g. After adjusting the pH of the resulting supernatant to 7.5, the sample was applied to Q-Sepharose FF (Amersham Pharmacia Biotech), equilibrated in 20 mm Tris, pH 7.5, and eluted with a NaCl gradient in equilibration buffer. α-Synuclein-containing fractions were identified by SDS-polyacrylamide gel electrophoresis and are >99% pure. The concentration of α-synuclein was determined by measuring absorbance at 280 nm and employing ε2800.1% of 0.354, determined by using Genetics Computer Group software. Purified samples of α-synuclein were concentrated to >7 mg/ml in Tris-buffered saline (20 mm Tris, pH 7.5, and 200 mm NaCl) + 0.05% sodium azide and sterile filtered through 0.22-μm filters to remove any particulate matter. The filtrates were all adjusted to a final concentration in the range of 2–7 mg/ml in Tris-buffered saline + 0.05% sodium azide and incubated at 37 °C in parafilm-sealed, 1.5-ml Beckman ultracentrifuge tubes. At various time points, the samples were centrifuged at 100,000 × g for 10 min, and the α-synuclein content of their supernatants was analyzed by measuring their absorbance at 280 nm. The concentration of α-synuclein was then determined employing ε2800.1% = 0.354. Supernatants of samples with concentrations 4 mg/ml or higher were first diluted 1:10 with Tris-buffered saline (11 μl of sample + 99 μl of buffer) whereas supernatants of samples at concentrations below 4 mg/ml were analyzed directly (100 μl). The remainder of the sample was vortexed for 30 s to resuspend pelleted material and then allowed to continue incubation at 37 °C. If the supernatant was analyzed neat, the 100-μl aliquot used for absorbance measurements was returned to the original incubation tube that was then vortexed for 30 s and placed back at 37 °C. Curve fits for aggregation time courses (i.e. A 280 versus time) were drawn manually. Solutions of wt or A53T α-synuclein at 7 mg/ml were incubated at 37 °C for 3 days in an Eppendorf Thermomixer with continuous shaking (high speed); under these conditions the equilibrium was reached. Reported seed concentrations are based on the amount of monomeric protein used, assuming complete aggregation of the starting material. The material was stored frozen at −20 °C until needed. Incubations of soluble α-synuclein at concentrations ranging from 2–7 mg/ml in Tris-buffered saline + 0.05% sodium azide were spiked with various amounts of preformed α-synuclein aggregates to serve as nuclei for fibril formation. The final concentration of seed is reported as a percentage of the soluble α-synuclein in the incubation (e.g. a 2 mg/ml incubation seeded at a level of 10% contains 0.2 mg/ml seed). Loss of soluble α-synuclein is measured byA 280 of soluble material following ultracentrifugation as described above. Critical concentrations were determined for wt and A53T mutant α-synuclein as described in Jarrett et al. (20Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1768) Google Scholar). α-Synuclein was incubated at 7 mg/ml in Tris-buffered saline, pH 7.5 + 0.05% sodium azide at 37 °C for 3 days with continuous shaking in an Eppendorf Thermomixer (high speed) to ensure complete aggregation. Following this treatment, the samples were centrifuged for 10 min at 100,000 ×g, the supernatants were collected, filtered through 0.22-μm filters and analyzed by quantitative amino acid analysis to determine protein content. Samples were transferred to pyrolyzed glass vial inserts, dried, and transferred to a Water's Picotag reaction vial, which contained 1 ml of a hydrolysis mixture (6n hydrochloric acid, 0.05% phenol, 0.001% β-mercaptoethanol). The reaction vial was purged with nitrogen and then sealed under vacuum; hydrolysis took place at 110 °C for 24 h. The sample glass inserts were removed and dried, and the samples were reconstituted in sample buffer containing the internal standard norleucine and analyzed on a Beckman 6300 amino acid analyzer (sodium format). Specialized software was used to calculate concentrations from the observed amino acids and internal standard recoveries. The software calculates a correlation coefficient and an average percentage difference between the observed and theoretical number of residues. To study the kinetics of α-synuclein fibrillogenesis the aggregated material was pelleted by centrifugation and the concentration of the remaining soluble material in the supernatant was determined (see “Experimental Procedures”). The loss of soluble material was paralleled by an increase in pellet size. In addition, we followed α-synuclein aggregation by turbidity measurements (data not shown) and showed earlier by electron and fluid phase atomic force microscopy that the light-scattering, sedimentable material is actually fibrillar (15Narhi 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. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). We have previously shown that α-synuclein forms fibrillar aggregatesin vitro and that this aggregation is preceded by a lag phase that is followed by a period of exponential aggregate formation (15Narhi 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. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar); both are monitored and pictured in Fig.1 as a loss of soluble wild type α-synuclein (filled symbols). This kinetic profile is indicative of a nucleated assembly process. In such a case, addition of exogenous nuclei (seeds) should start elongation instantly and thus expedite aggregation. Consistent with this prediction, the addition of preformed wild type α-synuclein aggregates did indeed seed soluble α-synuclein resulting in immediate aggregation as shown in Fig. 1 (open versus filled symbols). Seed concentrations in this experiment ranged from 0.001% () of the soluble α-synuclein amount up to 10% (⋄), and it is instantly apparent that the aggregation rate of soluble α-synuclein is controlled by the seed content in a dose-dependent manner. Analyzing the data from Fig. 1 as the time required to deplete 50% of the soluble starting material (t 12) allows a quantitative comparison of the seeding efficiency. At 2 mg/ml under our aggregation conditions (see “Experimental Procedures”) unseeded wild type α-synuclein (▪) aggregated with a half-life of 18 days. With the seed content consisting of only 0.001% () of the soluble α-synuclein, the t 12 already decreased to 16 days. At 0.01% (⊞) seed the t 12was 11 days and seed concentrations of 0.1 (Δ), 1 (○), and 10% (⋄) seed resulted in a further decrease of half-lives to 7, 4, and 0.5 days, respectively. Once the nuclei are present, either by de novo formation or exogenous addition, α-synuclein fibril elongation is predicted to proceed via the deposition of soluble α-synuclein units onto the existing templates provided by these nuclei. This process should be concentration-dependent with respect to the soluble α-synuclein concentration. We addressed this question by adding a fixed amount of exogenous, preformed seed (0.6 mg/ml) to incubations containing various concentrations of soluble α-synuclein (1–6 mg/ml) and followed its aggregation over time (Fig.2 A). In all tested concentrations the initial rates of soluble α-synuclein loss were linear or near linear until they started to level off, due to depletion of soluble material, and ultimately plateaued at the critical concentration; the rates decreased proportionally with decreasing α-synuclein concentration. In a seeded paradigm the rates of soluble α-synuclein loss are inversely proportional to the fiber elongation rates. Calculated initial elongation rates from this experiment are shown in Fig. 2 B as a function of soluble α-synuclein concentration. The linear correlation suggests that α-synuclein elongation is a first order process with respect to α-synuclein concentration. The experiments described above have addressed wild type α-synuclein aggregation. However, Fig.3 demonstrates that α-synuclein carrying the naturally occurring, dominant A53T mutation can also be homogeneously seeded (open versus filled symbols) and consequently also aggregates by a nucleation-dependent mechanism. As with wild type α-synuclein the rate of fibrillogenesis is seed concentration-dependent (○, 1%; ⋄, 10% seed). The dominant A53T α-synuclein mutation is associated with familial Parkinson's disease and co-expressed with the wild type form in mutation carriers. We have shown earlier that A53T as well as the only other known familial PD mutation displays accelerated aggregation and a significant reduction in the lag phase of this process. It was of immediate interest to test the hypothesis that the pathogenic mutant α-synuclein could act as heterogeneous seed for the soluble wild type protein. In Fig. 4 we added preformed aggregates of A53T α-synuclein to wild type α-synuclein and incubated under our standard aggregation conditions. Consistent with the data set shown in Fig. 1 the nonseeded control (▪) did not aggregate during the time course of this experiment. However, A53T seed concentrations comprising 1% (○) or 10% (⋄) of the soluble α-synuclein present in the incubation resulted in immediate fibril growth. The effect of heterogeneous seeding was, like in homogeneous seeding, clearly seed concentration-dependent: at 1% and 10% seed one-half of the soluble material was aggregated at 6 and 2 days, respectively. To determine the critical concentrations of α-synuclein proteins, wild type and A53T mutant preparations were incubated for 3 days at 37 °C and continuous shaking to ensure complete aggregation and equilibrium between fibers and soluble material. After centrifugation the supernatants were analyzed by quantitative amino acid analysis. The critical concentrations for wild type and A53T α-synuclein were 0.41 ± 0.01 mg/ml (28 μm) and 0.38 ± 0.09 mg/ml (26 μm), respectively, showing no statistically significant difference between these two isoforms. Lewy bodies that contain α-synuclein as their major fibrillar component are one of the hallmarks of Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. If and how Lewy bodies cause neuronal (or glial) degeneration is currently not known; however, the finding that two α-synuclein mutations cause autosomal dominant familial PD makes an α-synuclein cascade hypothesis attractive for PD. This hypothesis would assume that mutant α-synuclein would trigger the formation of Lewy bodies that in turn would cause the downstream PD pathology. Support for this model comes from recentin vitro studies, which demonstrate that purified full-length α-synuclein can indeed form fibrils similar to those found in Lewy bodies (11Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1271) Google Scholar, 13Hashimoto M. Hsu L. Sisk A. Xia Y. Takeda A. Sundsmo M. Masliah E. Brain Res. 1998; 799: 301-306Crossref PubMed Scopus (249) Google Scholar, 14Giasson B.I. Uryu K. Trojanowski J.Q. Lee V.M.-Y. J. Biol. Chem. 1999; 274: 7619-7622Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 15Narhi 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. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar) and which suggest aggregation-promoting conditions (23Hashimoto M. Hsu L. Xia Y. Takeda A. Sundsmo M. Masliah E. Neuroreport. 1999; 10: 1-5Crossref PubMed Scopus (168) Google Scholar). Most importantly, both mutations accelerate the formation of these fibrils (11Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1271) Google Scholar, 15Narhi 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. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Thus, the in vitro experiments mirror a critical aspect of the biology. The mechanism of this fibrillogenesis has not yet been addressed. In this study we show that α-synuclein aggregation follows a nucleation-elongation mechanism. Figs. 1 and 3 demonstrate that aggregation of both wild type and A53T mutant α-synuclein is rate-limited by a nucleation step. The addition of seeds, which act as nuclei, to aggregation-competent, supersaturated solutions of α-synuclein bypasses the lag phase and causes rapid aggregation. The aggregation rates are controlled by the seed content in a dose-dependent manner. By keeping the seed concentration constant and varying the soluble α-synuclein concentration we could show that its aggregation is also dependent on the soluble α-synuclein concentration (Fig. 2). The results suggest that α-synuclein fiber elongation is a first order process with respect to α-synuclein concentration. Interestingly, the elongation of amyloid β-peptide (Aβ) fibrils, which has been studied extensively by Maggio and colleagues (17Esler W.P. Stimson E.R. Ghilardi J.R. Vinters H.V. Lee J.P. Mantyh P.W. Maggio J.E. Biochemistry. 1996; 35: 749-757Crossref PubMed Scopus (124) Google Scholar, 24Esler W.P. Stimson E.R. Ghilardi J.R. Felix A.M. Lu Y.-A. Vinters H.V. Mantyh P.W. Maggio J.E. Nature Biotechnol. 1997; 15: 258-263Crossref PubMed Scopus (80) Google Scholar), follows first order kinetics, indicating that both Aβ and α-synuclein may follow the same general nucleation/elongation principles. We have determined the critical concentrations for wild type and A53T mutant forms of α-synuclein and found them to be indistinguishable within experimental error. This demonstrates that the enhanced aggregation tendency of the mutant forms is a kinetic effect and not due to an intrinsically lower solubility or higher fiber stability, respectively. It is tempting to speculate that the mutations lower the activation energy of critical regions of α-synuclein to convert from random coil to β-sheet, the conformation observed in the aggregate (15Narhi 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. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar), and that this conformation shift causes the rate-limiting nuclei to form. However, our results do not exclude the possibility that the nucleation of wild type and mutant α-synuclein forms could be mechanistically different. Also, additional factors may play a role as well as additional functional differences between wild type and mutant α-synuclein, e.g. α-synuclein vesicle binding (25Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, 26Jensen P.H. Nielsen M.S. Jakes R. Dotti C.G. Goedert M. J. Biol. Chem. 1998; 273: 26292-26294Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). It will be of interest to study the early events of nuclei formation and determine the nuclei size as well as the size and nature of potential intermediates. Such intermediates have been reported for different stages of other nucleated fibril formations, e.g.pre-nucleus dimers and post-nucleus protofibers in tau and amyloid β-peptide fibrillogenesis (18Friedhoff P. von Bergen M. Mandelkow E.-M. Davies P. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15712-15717Crossref PubMed Scopus (287) Google Scholar, 27Harper J.D. Wong S.S. Lieber C.M. Lansbury Jr., P.T. Chem. Biol. 1997; 4: 119-125Abstract Full Text PDF PubMed Scopus (631) Google Scholar, 28Walsh D.M. Lomakin A. Benedek G.B. Condron M.M. Teplow D.B. J. Biol. Chem. 1997; 272: 22364-22372Crossref PubMed Scopus (950) Google Scholar). Interestingly, Hashimotoet al. (13Hashimoto M. Hsu L. Sisk A. Xia Y. Takeda A. Sundsmo M. Masliah E. Brain Res. 1998; 799: 301-306Crossref PubMed Scopus (249) Google Scholar) already showed dimers and small oligomers of α-synuclein with SDS-polyacrylamide gel electrophoresis and suggested their time-dependent shift to high molecular, gel-excluded material. Our own and other (11Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1271) Google Scholar) preliminary observations utilizing among other methods fluid phase atomic force microscopy do indeed suggest the existence of other soluble α-synuclein species (data not shown). Most importantly, wild type α-synuclein cannot only be seeded by its own fibrils but can also be cross-seeded by mutant α-synuclein fibrils (Fig. 4) excluding significant structural differences at the elongation sites or growing tips of the two fiber types. This was intriguing and maybe even unexpected after recent studies reported structural differences in fibers of wild type and A53T mutant α-synuclein (14Giasson B.I. Uryu K. Trojanowski J.Q. Lee V.M.-Y. J. Biol. Chem. 1999; 274: 7619-7622Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar), and it will be interesting to study the ultrastructure of heterogeneous fibers. However, the fact that heterogeneous seeding is possible suggests that not only does mutant α-synuclein nucleate and consequently aggregate faster than wild type but that in a heterogeneous in vivo situation the lag phase of wild type α-synuclein aggregation is reduced to the one of the mutant protein; thus the mutant protein exerts its aggregation-accelerating effect on the total α-synuclein population. Based on this result we make the testable prediction that the Lewy bodies of mutation carriers will contain both the mutant and the wild type α-synuclein protein. It appears reasonable to hypothesize that under normal circumstances α-synuclein could show transient supersaturation in subcellular compartments of some neural cells. This transient supersaturation would not be harmful per se because individuals carrying the wild type α-synuclein allele would be protected from Lewy body formation by the long lag phase of the nucleation. However, α-synuclein mutations or unknown cellular factors could bypass the lag phase by increasing the rate of nuclei formation, enabling fibril growth and eventually causing their precipitation. In this scenario, α-synuclein nucleation would be at the core of Lewy body formation and nucleation inhibitors would, at least in principle, have therapeutic promise. We are grateful to Karen Sitney for the bacterial expression of recombinant proteins and to Scott Lauren for the quantitative amino acid analysis.