Conversion of Wild-type α-Synuclein into Mutant-type Fibrils and Its Propagation in the Presence of A30P Mutant
2009; Elsevier BV; Volume: 284; Issue: 12 Linguagem: Inglês
10.1074/jbc.m807482200
ISSN1083-351X
AutoresMotokuni Yonetani, Takashi Nonaka, M. Masuda, Yuki Inukai, Takayuki Oikawa, Shin‐ichi Hisanaga, Masato Hasegawa,
Tópico(s)Botulinum Toxin and Related Neurological Disorders
ResumoFibrillization or conformational change of α-synuclein is central in the pathogenesis of α-synucleinopathies, such as Parkinson disease. We found that the A30P mutant accelerates nucleation-dependent fibrillization of wild type (WT) α-synuclein. Electron microscopy observation and ultracentrifugation experiments revealed that shedding of fragments occurs from A30P fibrils and that these fragments accelerate fibrillization by serving as seeds. Immunochemical analysis using epitope-specific antibodies and biochemical analyses of protease-resistant cores demonstrated that A30P fibrils have a distinct conformation. Interestingly, WT fibrils formed with A30P seeds exhibited the same character as A30P fibrils, as did A30P fibrils formed with WT seeds, indicating that the A30P mutation affects the conformation and fibrillization of both WT and A30P. These effects of A30P mutation may explain the apparent conflict between the association of A30P with Parkinson disease and the slow fibrillization of A30P itself and therefore provide new insight into the molecular mechanisms of α-synucleinopathies. Fibrillization or conformational change of α-synuclein is central in the pathogenesis of α-synucleinopathies, such as Parkinson disease. We found that the A30P mutant accelerates nucleation-dependent fibrillization of wild type (WT) α-synuclein. Electron microscopy observation and ultracentrifugation experiments revealed that shedding of fragments occurs from A30P fibrils and that these fragments accelerate fibrillization by serving as seeds. Immunochemical analysis using epitope-specific antibodies and biochemical analyses of protease-resistant cores demonstrated that A30P fibrils have a distinct conformation. Interestingly, WT fibrils formed with A30P seeds exhibited the same character as A30P fibrils, as did A30P fibrils formed with WT seeds, indicating that the A30P mutation affects the conformation and fibrillization of both WT and A30P. These effects of A30P mutation may explain the apparent conflict between the association of A30P with Parkinson disease and the slow fibrillization of A30P itself and therefore provide new insight into the molecular mechanisms of α-synucleinopathies. Parkinson disease (PD) 2The abbreviations used are: PD, Parkinson disease; LB, Lewy body; WT, wild type; NAC, non-Aβ component of Alzheimer disease; Th-S, thioflavin S; MOPS, 3-(N-morpholino)propanesulfonic acid; CBB, Coomassie Brilliant Blue; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EM, electron microscopy. is the second most common neurodegenerative disorder, after Alzheimer disease. Neuropathological features of PD are selective loss of dopaminergic neurons in the substantia nigra and appearance of intracellular inclusion bodies, referred to as Lewy bodies (LBs) and Lewy neurites. Ultrastructurally, LBs are composed of a dense core of filamentous and granular material that is surrounded by radially oriented fibrils (1Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2443) Google Scholar, 2Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar). Biochemical and immunochemical analyses showed that hyperphosphorylated α-synuclein is the major component of the fibrous structures of LBs and Lewy neurites (3Fujiwara H. Hasegawa M. Dohmae N. Kawashima A. Masliah E. Goldberg M.S. Shen J. Takio K. Iwatsubo T. Nat. Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (162) Google Scholar). Genetic analyses of α-synuclein gene of familial cases of PD and dementia with LBs have demonstrated that expression of abnormal α-synuclein or overexpression of normal α-synuclein is associated with these diseases; namely, three missense mutations (A53T (4Polymeropoulos 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 Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6734) Google Scholar), A30P (5Kruger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schols L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3344) Google Scholar), and E46K (6Zarranz J.J. Alegre J. Gomez-Esteban J.C. Lezcano E. Ros R. Ampuero I. Vidal L. Hoenicka J. Rodriguez O. Atares B. Llorens V. Gomez Tortosa E. del Ser T. Munoz D.G. de Yebenes J.G. Ann. Neurol. 2004; 55: 164-173Crossref PubMed Scopus (2192) Google Scholar)) and multiplication (7Singleton A.B. Farrer M. Johnson J. Singleton A. Hague S. Kachergus J. Hulihan M. Peuralinna T. Dutra A. Nussbaum R. Lincoln S. Crawley A. Hanson M. Maraganore D. Adler C. Cookson M.R. Muenter M. Baptista M. Miller D. Blancato J. Hardy J. Gwinn-Hardy K. Science. 2003; 302: 841Crossref PubMed Scopus (3541) Google Scholar, 8Farrer M. Kachergus J. Forno L. Lincoln S. Wang D.S. Hulihan M. Maraganore D. Gwinn-Hardy K. Wszolek Z. Dickson D. Langston J.W. Ann. Neurol. 2004; 55: 174-179Crossref PubMed Scopus (594) Google Scholar, 9Chartier-Harlin M.C. Kachergus J. Roumier C. Mouroux V. Douay X. Lincoln S. Levecque C. Larvor L. Andrieux J. Hulihan M. Waucquier N. Defebvre L. Amouyel P. Farrer M. Destee A. Lancet. 2004; 364: 1167-1169Abstract Full Text Full Text PDF PubMed Scopus (1612) Google Scholar, 10Ibanez P. Bonnet A.M. Debarges B. Lohmann E. Tison F. Pollak P. Agid Y. Durr A. Brice A. Lancet. 2004; 364: 1169-1171Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 11Nishioka K. Hayashi S. Farrer M.J. Singleton A.B. Yoshino H. Imai H. Kitami T. Sato K. Kuroda R. Tomiyama H. Mizoguchi K. Murata M. Toda T. Imoto I. Inazawa J. Mizuno Y. Hattori N. Ann. Neurol. 2006; 59: 298-309Crossref PubMed Scopus (258) Google Scholar, 12Fuchs J. Nilsson C. Kachergus J. Munz M. Larsson E.M. Schule B. Langston J.W. Middleton F.A. Ross O.A. Hulihan M. Gasser T. Farrer M.J. Neurology. 2007; 68: 916-922Crossref PubMed Scopus (310) Google Scholar) of the α-synuclein gene have been found to cosegregate with the onset of PD in kindreds of autosomal dominantly inherited familial PD and dementia with LBs. α-Synuclein is a 140-amino acid protein, harboring seven imperfect tandem repeats (KTKEGV-type) in the N-terminal half, followed by a hydrophobic central region (non-Aβ component of Alzheimer disease (NAC)) and an acidic C-terminal. The tandem repeat region has been assumed to form an amphipathic α-helix by binding to phospholipid (13Davidson 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). Circular dichroism and Fourier-transform IR analysis revealed that α-synuclein is a natively unfolded protein with little ordered secondary structure (14Uversky V.N. Li J. Fink A.L. J. Biol. Chem. 2001; 276: 10737-10744Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). However, recent NMR analyses have revealed three intramolecular long range interactions. These interactions are between the highly hydrophobic NAC region (residues 85-95) and the C terminus (residues 110-130), C-terminal residues 120-130 and residues 105-115, and the region around residue 120 and the N terminus around residue 20 (15Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (637) Google Scholar). Recombinant α-synuclein in vitro assembles into fibrils that closely resemble those in brains with PD and dementia with LBs upon incubation at a high concentration at 37 °C with shaking, whereas other synuclein family proteins (i.e. β-synuclein and γ-synuclein) neither accumulate in the brain (1Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2443) Google Scholar, 16Spillantini M.G. Schmidt M.L. Lee V.M. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6267) Google Scholar) nor form fibrils (17Serpell L.C. Berriman J. Jakes R. Goedert M. Crowther R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4897-4902Crossref PubMed Scopus (661) Google Scholar, 18Crowther R.A. Daniel S.E. Goedert M. Neurosci. Lett. 2000; 292: 128-130Crossref PubMed Scopus (121) Google Scholar, 19Biere 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. Citron M. J. Biol. Chem. 2000; 275: 34574-34579Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). During the assembly of α-synuclein fibrils, conformational change from random coil to β-sheet structure can be observed. It has been shown that the sequence of the NAC region in α-synuclein is necessary for the assembly (20Giasson B.I. Murray I.V. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2001; 276: 2380-2386Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar). Mostly in in vitro experiments, it has been shown that the A53T and E46K mutations promote fibrillization (17Serpell L.C. Berriman J. Jakes R. Goedert M. Crowther R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4897-4902Crossref PubMed Scopus (661) Google Scholar, 21Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1271) Google Scholar, 22Narhi 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, 23Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1347) Google Scholar, 24Conway K.A. Harper J.D. Lansbury Jr., P.T. Biochemistry. 2000; 39: 2552-2563Crossref PubMed Scopus (695) Google Scholar, 25Greenbaum E.A. Graves C.L. Mishizen-Eberz A.J. Lupoli M.A. Lynch D.R. Englander S.W. Axelsen P.H. Giasson B.I. J. Biol. Chem. 2005; 280: 7800-7807Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), whereas the effect of A30P mutation on fibrillization is unclear. It has been reported that A30P mutation promotes oligomerization of nonfibrillar protofibrils (23Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1347) Google Scholar, 26Goldberg M.S. Lansbury Jr., P.T. Nat. Cell Biol. 2000; 2: E115-E119Crossref PubMed Scopus (453) Google Scholar) and that some of the protofibrils with a circular morphology may form pores by binding to ER membrane (27Volles M.J. Lansbury Jr., P.T. Biochemistry. 2002; 41: 4595-4602Crossref PubMed Scopus (426) Google Scholar). It has also been reported that A30P mutation is defective in binding to phospholipid vesicles, and the alteration of membrane interaction could contribute to early onset of PD (28Jensen 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, 29Jo E. Fuller N. Rand R.P. St George-Hyslop P. Fraser P.E. J. Mol. Biol. 2002; 315: 799-807Crossref PubMed Scopus (187) Google Scholar). Assembly of protein into fibrils is usually a nucleation-dependent process that consists of a lag phase (nucleation) and a growth phase (elongation). α-Synuclein fibrillization was confirmed to be a nucleation-dependent process (22Narhi 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). The addition of seeds to the monomer promotes fibrillization by rendering the nucleation process redundant. Not only wild type (WT) fibrils but also A53T fibrils have been reported to act as nuclei for fibrillization of WT α-synuclein (30Wood S.J. Wypych J. Steavenson S. Louis J.C. Citron M. Biere A.L. J. Biol. Chem. 1999; 274: 19509-19512Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar). In this study, we have investigated nucleation-dependent fibrillization of WT and A30P α-synuclein and the conformations of WT and A30P fibrils formed in the presence of WT and A30P seeds. We found that A30P seeds accelerated the nucleation-dependent fibrillization of WT α-synuclein more effectively than did WT seeds. Further, A30P fibrils have a distinct conformation from WT fibrils and show a higher level of fragment shedding. The WT fibrils formed in the presence of A30P seeds showed the same character as A30P fibrils, suggesting that the nucleation-dependent assembly of WT fibrils in the presence of A30P seeds results in conversion of WT conformation to that of A30P. Further, the A30P fibrils formed in the presence of WT seeds shared the properties of A30P fibrils. The in vitro results shown here implicate the structural and functional differences among α-synuclein amyloid fibrils, useful for understanding the pathogenesis of α-synucleinopathies. Antibodies-α-Synuclein epitope-specific polyclonal antibodies syn1-10, syn75-91, and syn131-140 were raised against synthetic peptides MDVFMKGLSKC (residues 1-10 with Cys at the C terminus), CTAVAQKTVEGAGSIAAA (residues 75-91 with Cys at the N terminus), and CEGYQDYEPEA (residues 131-140 with Cys at the N terminus) of human α-synuclein, respectively. Peptides were conjugated to m-maleimidobenzoyl-N-hydrosuccinimide ester-activated keyhole limpet hemocyanin. The keyhole limpet hemocyanin-peptide complex (1 mg of each immunogen) emulsified in Freund's complete adjuvant was injected subcutaneously into a New Zealand white rabbit, followed by five weekly subcutaneous injections of 150 mg of KLH-peptide complex emulsified in Freund's incomplete adjuvant starting from 3 weeks after the first immunization. Other anti-α-synuclein antibodies, number 36 (residues 1-10) and NAC2 (residues 75-91), were kindly provided by Dr. Iwatsubo and Dr. Jäkälä, respectively. Expression and Purification of Human WT and Mutant α-Synuclein-Human α-synuclein cDNA in bacterial expression plasmid pRK172 was provided by Dr. Goedert. A30P, E46K, and A53T mutations were induced by site-directed mutagenesis (Stratagene). WT and mutant α-synuclein were expressed in Escherichia coli BL21 (DE3) cells and purified as described (31Masuda M. Dohmae N. Nonaka T. Oikawa T. Hisanaga S. Goedert M. Hasegawa M. FEBS Lett. 2006; 580: 1775-1779Crossref PubMed Scopus (65) Google Scholar). Protein concentration was determined as described (31Masuda M. Dohmae N. Nonaka T. Oikawa T. Hisanaga S. Goedert M. Hasegawa M. FEBS Lett. 2006; 580: 1775-1779Crossref PubMed Scopus (65) Google Scholar). Fibrillization of WT and Mutant α-Synuclein-Purified WT and mutant α-synuclein (1 mg/ml) were each incubated at 37 °C, with shaking at 200 rpm in 30 mm Tris-HCl, pH 7.5, containing 0.1% NaN3. For quantitative assessment of fibrillization, aliquots (10 μl) of assembly mixture were removed at various time points, brought to 300 μl with 5 mm Thioflavin S (Th-S) in 20 mm MOPS, pH 6.8, and incubated for 60 min at room temperature. Fluorimetry was performed using a Hitachi F4000 fluorescence spectrophotometer (set at 440 nm excitation/521 nm emission) as described (32Taniguchi S. Suzuki N. Masuda M. Hisanaga S. Iwatsubo T. Goedert M. Hasegawa M. J. Biol. Chem. 2005; 280: 7614-7623Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Preparation of Seeds-Purified WT and mutant α-synuclein (7 mg/ml) were each incubated for 96-120 h at 37 °C, with shaking at 200 rpm in 30 mm Tris-HCl, pH 7.5, containing 0.1% NaN3. Assembly mixture was diluted in 5 volumes of 30 mm Tris-HCl, pH 7.5, and ultracentrifuged at 151,000 × g for 20 min at 25 °C. The pellets were resuspended in 5 volumes of 30 mm Tris-HCl, pH 7.5, and ultracentrifuged at 151,000 × g for 20 min again. The pellets were resuspended homogeneously by pipetting in 30 mm Tris-HCl, pH 7.5, containing 0.1% NaN3 and used as seeds. Aliquots of seeds or fibrils were solubilized in 6 m guanidine hydrochloride, and the concentration of α-synuclein was determined as described (31Masuda M. Dohmae N. Nonaka T. Oikawa T. Hisanaga S. Goedert M. Hasegawa M. FEBS Lett. 2006; 580: 1775-1779Crossref PubMed Scopus (65) Google Scholar). Nucleation-dependent Fibrillization of α-Synuclein-Purified WT or mutant α-synuclein (1 mg/ml) in 30 mm Tris-HCl, pH 7.5, containing 0.1% NaN3 was incubated with seeds (1% of total protein) for 0-144 h at 37 °C without shaking. Fibrillization was monitored by measuring Th-S fluorescence. Semiquantitative Analysis of Fibril Length in Suspended Fibrils-Fibrils formed in the presence or absence of seeds (0.1 mg/ml) were observed at a magnification of ×25,000 by electron microscopy after suspension by pipetting. Fibril length was measured on the photographs, and the populations were calculated. Characterization of Seeds by Ultracentrifugation-Seeds or fibrils formed in the presence of seeds (1.0 mg/ml) were suspended in 5 volumes of 30 mm Tris-HCl, pH 7.5, and incubated for 30 min at room temperature, followed by ultracentrifugation for 20 min at 109,000 × g. The pellets were resuspended in equal volumes of the supernatant with 30 mm Tris-HCl, pH 7.5. The supernatant and suspension were treated with 5× SDS sample buffer and subjected to SDS-PAGE. After staining of gels with Coomassie Brilliant Blue (CBB) and scanning, the intensities of the α-synuclein band were quantified by Scion Image (Scion Corp.). Aliquots of the supernatants were examined by electron microscopy and used for studies of nucleation-dependent fibrillization. Electron Microscopy-Fibrils, seeds and fibrils formed in the presence of seeds were diluted in 30 mm Tris-HCl, pH 7.5. Aliquots of these dilutions and centrifugal supernatants of seeds and fibrils formed in the presence of seeds were placed on 400-mesh collodion-coated grids, negatively stained with 2% lithium phosphotungstate, and observed with a JEOL 1200EXII electron microscope. Dot Blot Assay-α-Synuclein monomer, seeds and fibrils formed in the presence of seeds were diluted in 30 mm Tris-HCl, pH 7.5, 0.1% NaN3 and spotted onto polyvinylidene difluoride membrane. The membrane was probed with epitope-specific α-synuclein antibodies syn1-10 (N-terminal region), syn75-91 (NAC region), and syn131-140 (C-terminal region) or stained with CBB to detect total protein. Immunoreactivity was visualized using the avidin-biotin detection system (Vector Laboratories) and quantified by scanning as described above. Comparison of Protease-resistant Cores of α-Synuclein Fibrils-Seeds or fibrils formed in the presence of seeds (1.0 mg/ml) were sonicated and treated with 50 μg/ml trypsin or 2 μg/ml proteinase K at 37 °C for 30 min. The reaction was stopped by boiling for 5 min. The solution was treated with sample buffer containing 2% SDS and 8 m urea and subjected to SDS-PAGE. Cytotoxicity Assay-The cytotoxic effect of α-synuclein fibrils was assessed by measuring cellular redox activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as described (33Masuda M. Suzuki N. Taniguchi S. Oikawa T. Nonaka T. Iwatsubo T. Hisanaga S. Goedert M. Hasegawa M. Biochemistry. 2006; 45: 6085-6094Crossref PubMed Scopus (321) Google Scholar). Briefly, SH-SY5Y cells cultured in a 96-well microtiter plate were treated with 500 nm α-synuclein monomer, fibrils (suspended by pipetting or sonication), or fibrils formed in the presence of seeds. Following a 6-h incubation, the cytotoxic effect was assessed by measuring cellular redox activity. Statistical Analysis-Statistic analysis was performed using unpaired Student's t test. The results are expressed as means ± S.E. of three independent experiments (n = 3). Effect of A30P Seeds on Fibrillization of α-Synuclein-First, we tested the effect of mutations on fibrillization of α-synuclein. As shown in Fig. 1A, both A53T and E46K mutants fibrillized faster than WT, whereas the fibrillization of A30P mutant was much slower than that of WT, confirming the previous observations. Since A53T fibrils can act as seeds for the fibrillization of WT α-synuclein (30Wood S.J. Wypych J. Steavenson S. Louis J.C. Citron M. Biere A.L. J. Biol. Chem. 1999; 274: 19509-19512Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar), we next investigated whether A30P fibrils also act as seeds for WT monomer. We incubated WT α-synuclein with WT, A30P, E46K, or A53T seeds under conditions where WT α-synuclein itself does not form fibrils and analyzed fibrillization (Fig. 1B). Fibrillization was observed following the addition of either WT seeds or mutant seeds. Interestingly, the assembly was faster in the presence of A30P seeds than in the presence of WT or the other mutant seeds. The time required for half-maximal fibrillization was ∼1.5 days with A30P seeds, which is shorter than those with the other seeds, WT (2.5 days), E46K (more than 6 days), and A53T (6 days). In the brains of patients with the A30P mutation, both WT and A30P α-synuclein are expressed. Therefore, a mixture of WT and A30P α-synuclein was incubated with WT or A30P seeds, and the progress of fibrillization was observed in terms of Th-S fluorescence intensity. As in the case of WT α-synuclein monomer alone, A30P seeds accelerated the fibrillization process more effectively than did WT seeds (supplemental Fig. 1). Fibrillization was not observed in the absence of seeds. The time for half-maximal fibrillization was ∼1.5 days with A30P seeds and 3.5 days with WT seeds. Shedding of Fragments from A30P Fibrils-To elucidate the mechanism of the effect of A30P seeds on fibrillization, we utilized electron microscopy (Fig. 2). Many tiny or short fibrils were observed in A30P seeds, whereas relatively long fibrils were predominantly detected in WT, E46K, and A53T seeds (Fig. 2A, v, vi, vii, and viii). This observation was confirmed by measuring the fibril length in these seeds. Short fibrils of less than 100 nm were predominant in the A30P seeds, whereas longer fibrils were detected in the WT and A53T seeds (supplemental Fig. 3A). Before preparation of the seeds, WT and mutant α-synuclein fibrils were uniformly long and showed no morphological differences (Fig. 2A, i, ii, iii, and iv). Therefore, it appeared that A30P fibrils readily fragmented during the process of seed preparation. To test whether the small fibrils could be separated by centrifugation or not, WT and mutant α-synuclein seeds were ultracentrifuged, and the supernatants were observed by electron microscopy. Surprisingly, many tiny fibrils were observed in the supernatant of A30P seeds, whereas such tiny fibrils were hardly detected in the supernatant of WT, E46K, and A53T seeds (Fig. 2A, ix, x, xi, and xii), indicating that A30P fibrils have a higher propensity for shedding fragments than do WT fibrils and that the small fragments are recovered in the supernatant of ultracentrifugation. We then attempted to quantitate the fragmented fibrils by ultracentrifugation. The centrifugal supernatant was subjected to SDS-PAGE, and the gel was stained with CBB. As expected, more α-synuclein was detected in the supernatant of A30P seeds than in those of WT, E46K, and A53T seeds (Fig. 2, B and C). Next, we investigated whether the tiny fibrils recovered in the supernatant of ultracentrifugation can act as seeds for fibrillization of α-synuclein. WT α-synuclein was incubated with the supernatant of WT, A30P, E46K, or A53T seeds (10% of total volume), and fibrillization was monitored by a Th-S assay. In the presence of the supernatant of WT, E46K, and A53T seeds, a very slow increase of Th-S was observed, whereas fibrillization was accelerated in the presence of the supernatant of A30P seeds (Fig. 2D), as seen in the fibrillization in Fig. 1B. These results indicate that the A30P fibrils readily shed many tiny fibrils that can act as seeds for fibrillization. Shedding Property of WT Fibrils Formed in the Presence of A30P Seeds-Next, we investigated whether or not the WT fibrils formed in the presence of A30P seeds have the same properties as A30P fibrils (Fig. 3A). The WT fibrils formed in the presence of A30P seeds appeared to be morphologically indistinguishable from the WT fibrils formed in the presence of WT seeds before preparation of suspensions (Fig. 3A, i and ii). However, after suspension, many tiny fibrils were shed from WT fibrils formed in the presence of A30P seeds, whereas only a few short fibrils were shed from WT fibrils formed in the presence of WT seeds (Fig. 3A, iii and iv). This was confirmed by measuring the fibril length in these suspensions of fibrils produced by pipetting (supplemental Fig. 3B). After ultracentrifugation, many tiny fibrils were observed in the supernatant of WT fibrils formed in the presence of A30P seeds, whereas few such fibrils were detected in the supernatant of WT fibrils formed in the presence of WT seeds (Fig. 3A, v and vi). Quantitative analysis confirmed that a larger amount of α-synuclein was present in the supernatant of WT fibrils formed in the presence of A30P seeds than in that of WT fibrils formed in the presence of WT seeds (Fig. 3, B and C). These results suggest that WT fibrils formed in the presence of A30P seeds have the same shedding propensity as A30P fibrils. Immunochemical Analysis of α-Synuclein Fibrils with Epitope-specific Antibodies-To investigate the structural differences between WT fibrils and A30P fibrils and between WT fibrils formed in the presence of WT seeds and WT fibrils formed in the presence of A30P seeds, we employed a dot blot assay with three epitope-specific antibodies to α-synuclein, syn1-10 (N-terminal region), syn75-91 (NAC region), and syn131-140 (C-terminal region). As shown in Fig. 4, G and I, syn131-140 stained both WT and A30P fibrils (fibril seeds) almost equally, whereas syn75-91 (Fig. 4, E and I) strongly labeled only WT fibrils. syn1-10 (Fig. 4, C and I) labeled A30P fibrils more strongly than WT fibrils. Similar results were obtained with other independently produced anti-α-synuclein antibodies to the N terminus (number 36, a gift from Dr. Iwatsubo) and anti-NAC antibody (NAC2, a gift form Dr. Jäkälä) (data not shown). These results suggest that the conformation of A30P fibrils is different from that of WT fibrils. Interestingly, dot blot analysis of WT fibrils formed in the presence of A30P seeds showed a pattern of immunoreactivity similar to that of A30P fibrils (Fig. 4, D, F, and I), whereas WT fibrils formed in the presence of WT seeds showed the same pattern as WT fibrils (Fig. 4, D, F, and I). These results suggest that the WT fibrils formed in the presence of A30P seeds have a similar conformation to that of A30P fibrils. WT or A30P monomer showed very weak immunoreactivities to syn1-10 and syn75-91 (supplemental Fig. 2, C, E, and I). WT fibrils showed comparatively strong immunoreactivities to all three antibodies, whereas A30P fibrils showed a distinct pattern (supplemental Fig. 2, D, F, and I), being labeled strongly with syn1-10 but hardly at all with syn75-91. Comparison of Protease-resistant Cores of WT and A30P α-Synuclein Fibrils-In order to investigate the structural differences between WT and A30P fibrils and also between WT fibrils formed in the presence of WT seeds and WT fibrils formed in the presence of A30P seeds, we analyzed protease-resistant cores of these fibrils after digestion with trypsin or proteinase K. Amyloid fibrils in neurodegenerative diseases and other types of amyloidosis are known to be highly resistant to many proteases, and the analysis of protease-resistant cores is frequently used for investigating the structures of various amyloid fibrils (34Miake H. Mizusawa H. Iwatsubo T. Hasegawa M. J. Biol. Chem. 2002; 277: 19213-19219Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 35Aoyagi H. Hasegawa M. Tamaoka A. J. Biol. Chem. 2007; 282: 20309-20318Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 36Raymond G.J. Hope J. Kocisko D.A. Priola S.A. Raymond L.D. Bossers A. Ironside J. Will R.G. Chen S.G. Petersen R.B. Gambetti P. Rubenstein R. Smits M.A. Lansbury Jr., P.T. Caughey B. Nature. 1997; 388: 285-288Crossref PubMed Scopus (239) Google Scholar). When monomeric α-synuclein was digested with trypsin or proteinase K, no band was detected (Fig. 5A). In contrast, 8-12 kDa core bands remained when the fibrils were treated with trypsin or proteinase K. Trypsin digestion of the fibrils composed of WT afforded two major bands of 10 and ∼13 kDa (black arrowheads), and a similar band pattern was observed after the digestion of the WT fibrils formed in the presence of the WT seeds. On the other hand, two major bands of 9.5 and ∼12.5 kDa (white arrowheads) with smaller molecular weights than those of the WT bands were detected after the digestion of A30P fibrils and WT fibrils formed in the presence of A30P seeds (Fig. 5, B and C). Similarly, digestion of WT fibrils and WT fibrils formed in the presence of WT seeds with proteinase K, a nonspecific protease, showed three major bands of 10-12 kDa (black arrowheads), whereas one major band of ∼11.5 kDa (white arrowhead) was detected after the digestion of A30P fibrils and WT fibrils formed in the presence of A30P seeds (Fig. 5, B and C). These protein-chemical data strongly suggest that the core structures of A30P fibrils and WT fibrils formed in the presence of A30P seeds are distinct from those of WT fibrils and WT fibrils formed in the presence of WT seeds and further support the immunochemical results described above. Effect of WT Seeds on Fibrillization of A30P Mutant α-Synuclein-Since WT α-synuclein assembles into fibrils fast
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