Conversion of Wild-type α-Synuclein into Mutant-type Fibrils and Its Propagation in the Presence of A30P Mutant

Abstract

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. 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: Full Text Full Text PDF PubMed Scopus Google Scholar). the of α-synuclein fibrils, conformational change from to structure has been that the of the NAC region in α-synuclein is the Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). in in vitro has been that the and E46K mutations 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, Nat. 1998; 4: PubMed Scopus Google Scholar, L. Wood S.J. Steavenson S. Jiang Y. Wu G.M. Anafi D. Martin F. K. P. Louis J.C. Wypych J. A.L. Citron M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Lee S.J. J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar, M.A. P.H. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), whereas the of A30P mutation fibrillization is has been that A30P mutation of Lee S.J. J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar, M.S. Nat. Cell Biol. 2000; PubMed Scopus Google Scholar) and that of the with a may form by binding to M.J. 2002; PubMed Scopus Google Scholar). has been that A30P mutation is in binding to phospholipid and the of to onset of PD P.H. M.S. Jakes R. Goedert M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, E. N. P. J. Biol. 2002; PubMed Scopus Google Scholar). of protein into fibrils is a nucleation-dependent that of a and a α-Synuclein fibrillization to a nucleation-dependent L. Wood S.J. Steavenson S. Jiang Y. Wu G.M. Anafi D. Martin F. K. P. Louis J.C. Wypych J. A.L. Citron M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of seeds to the fibrillization by the wild type (WT) fibrils fibrils have been to as fibrillization of WT α-synuclein S.J. Wypych J. Steavenson S. Louis J.C. Citron M. A.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have nucleation-dependent fibrillization of WT and A30P α-synuclein and the of WT and A30P fibrils formed in the of WT and A30P seeds. We found that A30P seeds the nucleation-dependent fibrillization of WT α-synuclein did WT seeds. A30P fibrils have a distinct conformation from WT fibrils and a of The WT fibrils formed in the of A30P seeds showed the same character as A30P fibrils, that the nucleation-dependent of WT fibrils in the of A30P seeds in of WT conformation to that of A30P. the A30P fibrils formed in the of WT seeds the of A30P The in vitro the and α-synuclein fibrils, the pathogenesis of α-synucleinopathies. epitope-specific antibodies and (residues with at the C (residues with at the N and (residues with at the N of to The of in into a followed by of of in from after the (residues and (residues by Iwatsubo and and of WT and α-synuclein in expression by and mutations by WT and mutant α-synuclein in and as M. Dohmae N. T. T. S. Goedert M. Hasegawa M. Lett. 2006; PubMed Scopus Google Scholar). concentration as M. Dohmae N. T. T. S. Goedert M. Hasegawa M. Lett. 2006; PubMed Scopus Google Scholar). Fibrillization of WT and WT and mutant α-synuclein at 37 with at in of of at to with in 20 MOPS, and at using a at as S. N. M. S. Iwatsubo T. Goedert M. Hasegawa M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of WT and mutant α-synuclein at 37 with at in in of and at 20 at The in of and at 20 The by in and used as seeds. of seeds or fibrils in and the concentration of α-synuclein as M. Dohmae N. T. T. S. Goedert M. Hasegawa M. Lett. 2006; PubMed Scopus Google Scholar). Fibrillization of WT or mutant α-synuclein in with seeds of at 37 °C Fibrillization by of in formed in the or of seeds at a of by electron microscopy after by the and the of by or fibrils formed in the of seeds in of and at followed by ultracentrifugation 20 at The in of the with The and with and to of with Coomassie Brilliant and the of the α-synuclein by of the by electron microscopy and used of nucleation-dependent Electron seeds and fibrils formed in the of seeds in of these and of seeds and fibrils formed in the of seeds with and with a electron seeds and fibrils formed in the of seeds in and The with epitope-specific α-synuclein antibodies and or with to using the and by as of of α-Synuclein or fibrils formed in the of seeds and with or at 37 °C The by The with and and to of α-synuclein fibrils by with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium as M. N. S. T. T. Iwatsubo T. S. Goedert M. Hasegawa M. 2006; PubMed Scopus Google Scholar). in a with α-synuclein fibrils by or or fibrils formed in the of seeds. a the by analysis using The are as S.E. of three experiments of A30P Fibrillization of the of mutations fibrillization of α-synuclein. in both and E46K WT, whereas the fibrillization of A30P mutant that of WT, the fibrils as seeds the fibrillization of WT α-synuclein S.J. Wypych J. Steavenson S. Louis J.C. Citron M. A.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), A30P fibrils as seeds WT We WT α-synuclein with WT, or seeds WT α-synuclein itself form fibrils and fibrillization Fibrillization the of WT seeds or mutant seeds. Interestingly, the in the of A30P seeds in the of WT or the other mutant seeds. The fibrillization with A30P seeds, is those with the other seeds, WT E46K and the brains of with the A30P both WT and A30P α-synuclein are a of WT and A30P α-synuclein with WT or A30P seeds, and the of fibrillization in of in the of WT α-synuclein A30P seeds the fibrillization did WT seeds Fibrillization in the of seeds. The fibrillization with A30P seeds and with WT seeds. of from A30P the of the of A30P seeds electron microscopy or fibrils in A30P seeds, whereas long fibrils in WT, and seeds and observation by the in these seeds. fibrils of in the A30P seeds, whereas fibrils in the WT and seeds of the seeds, WT and mutant α-synuclein fibrils long and showed and that A30P fibrils the of the fibrils by or WT and mutant α-synuclein seeds and the by electron microscopy. fibrils in the of A30P seeds, whereas such fibrils in the of WT, and seeds and indicating that A30P fibrils have a shedding fragments WT fibrils and that the fragments are in the of We to the fibrils by The to and the with α-synuclein in the of A30P seeds in those of WT, and seeds and the fibrils in the of ultracentrifugation as seeds fibrillization of α-synuclein. WT α-synuclein with the of WT, or seeds of and fibrillization by a the of the of WT, and seeds, a slow of whereas fibrillization in the of the of A30P seeds as in the fibrillization in These that the A30P fibrils fibrils that as seeds of WT in the of A30P or the WT fibrils formed in the of A30P seeds have the same as A30P fibrils The WT fibrils formed in the of A30P seeds to from the WT fibrils formed in the of WT seeds of and However, after fibrils from WT fibrils formed in the of A30P seeds, whereas a fibrils from WT fibrils formed in the of WT seeds and by the in these of fibrils by fibrils in the of WT fibrils formed in the of A30P seeds, whereas such fibrils in the of WT fibrils formed in the of WT seeds and analysis that a of α-synuclein in the of WT fibrils formed in the of A30P seeds in that of WT fibrils formed in the of WT seeds and These that WT fibrils formed in the of A30P seeds have the same shedding as A30P Immunochemical of α-Synuclein with the between WT fibrils and A30P fibrils and between WT fibrils formed in the of WT seeds and WT fibrils formed in the of A30P seeds, a with three epitope-specific antibodies to and in and both WT and A30P fibrils whereas and WT C and A30P fibrils WT with other antibodies to the N terminus a from and a form These that the conformation of A30P fibrils is from that of WT Interestingly, analysis of WT fibrils formed in the of A30P seeds showed a of to that of A30P fibrils and whereas WT fibrils formed in the of WT seeds showed the same as WT fibrils and These that the WT fibrils formed in the of A30P seeds have a conformation to that of A30P WT or A30P showed to and and WT fibrils showed to three whereas A30P fibrils showed a distinct and with at with of of WT and A30P α-Synuclein to the between WT and A30P fibrils and between WT fibrils formed in the of WT seeds and WT fibrils formed in the of A30P seeds, protease-resistant cores of these fibrils after with or K. fibrils in neurodegenerative and other of are to highly to and the analysis of protease-resistant cores is used the structures of fibrils H. H. Iwatsubo T. Hasegawa M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, H. Hasegawa M. A. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. J. P. Rubenstein R. M.A. B. Nature. 1997; 388: PubMed Scopus Google Scholar). α-synuclein with or core the fibrils with or K. of the fibrils composed of WT major of and and a after the of the WT fibrils formed in the of the WT seeds. the other major of and with molecular those of the WT after the of A30P fibrils and WT fibrils formed in the of A30P seeds and of WT fibrils and WT fibrils formed in the of WT seeds with a showed three major of whereas major of after the of A30P fibrils and WT fibrils formed in the of A30P seeds and These that the core structures of A30P fibrils and WT fibrils formed in the of A30P seeds are distinct from those of WT fibrils and WT fibrils formed in the of WT seeds and the immunochemical of WT Fibrillization of A30P WT α-synuclein assembles into fibrils A30P mutant α-synuclein in vitro WT seeds may formed A30P seeds in the brains of with the A30P We therefore A30P mutant α-synuclein into fibrils in the of WT seeds and the A30P fibrils formed in the of WT seeds the of WT fibrillization of A30P mutant α-synuclein in the of WT seeds. the A30P fibrils formed in the of WT seeds by electron microscopy and ultracentrifugation the A30P fibrils formed in the of WT seeds to from the WT fibrils formed in the of WT seeds the of and However, after fibrils in the A30P fibrils formed in the of WT seeds, whereas such fibrils in the WT fibrils formed in the of WT seeds and by the of fibrils in these by fibrils in the of A30P fibrils formed in the of WT seeds, whereas such fibrils in the of WT fibrils formed in the of WT seeds and analysis of α-synuclein in the and by the of α-synuclein in the of A30P fibrils formed in the of WT seeds in that of WT fibrils formed in the of WT seeds and These that A30P fibrils formed in the of WT seeds the character of the WT seeds the character of A30P of A30P in the of WT the between the shedding of fibrils and A30P fibrils formed in the of WT seeds by using the epitope-specific antibodies A30P fibrils formed in the of WT seeds showed the same of as A30P fibrils, and the from that of WT fibrils formed in the of WT seeds and with and antibodies the between WT fibrils formed in the of WT seeds and A30P fibrils formed in the of WT seeds, the protease-resistant cores of the fibrils in and the of the and cores of A30P fibrils formed in the of WT seeds the same as those of A30P fibrils and distinct from those of WT fibrils and WT fibrils formed with WT seeds These the that A30P fibrils formed in the of WT seeds the conformation of the WT seeds the conformation of A30P of WT and α-Synuclein the between the shedding of fibrils and the with WT or mutant fibrils by or and the by an as has been used in fibrils composed of WT or mutant α-synuclein showed a of whereas with WT or mutant α-synuclein. Interestingly, A30P fibrils showed a WT fibrils or the other mutant fibrils, a between shedding and with fibrils, to fibrils the the in the of the E46K mutant These that of fibrils are of long is that fibrils with long The of WT fibrils formed in the of A30P seeds and A30P fibrils formed in the of WT seeds by These fibrils with shedding showed WT fibrils formed in the of WT seeds, and the effects of these These a between the shedding of fibrils and the has been to a in the fibrillization of protein, and H. K. K. T. Google Scholar, J.T. Full Text PDF PubMed Scopus Google Scholar, H. F. PubMed Scopus Google Scholar). of α-synuclein into fibrils has been to a nucleation-dependent L. Wood S.J. Steavenson S. Jiang Y. Wu G.M. Anafi D. Martin F. K. P. Louis J.C. Wypych J. A.L. Citron M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have the nucleation-dependent of WT and mutant α-synuclein into fibrils, the shedding of these fibrils, and the conformational between WT and A30P fibrils and between these fibrils formed in the of seeds. The of A30P seeds to WT the fibrillization of WT indicating that A30P fibrils have a WT α-synuclein. A30P seeds the fibrillization of WT did WT, and seeds. the of seeds WT α-synuclein to that of WT seeds, and that of E46K seeds and ultracentrifugation revealed that the of A30P to the shedding of A30P of fibrils has been found in of protein M. Nature. 2006; PubMed Scopus Google Scholar). N-terminal residues into fibrils with or upon incubation at or 37 a and high and fibrillization of has been that protein by of protein with B. C. Nature. 2001; PubMed Scopus Google Scholar). These that or shedding of fibrils accelerate fibrillization and in high and are therefore with of to at residue may have a the conformation of α-synuclein and a in the α-helix structure and has been that in the A30P a region of structure (residues that in WT is of structure is and the of is J.C. R. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar, R. D. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). the A30P mutation in the of the N terminus of the core (residues H. H. Iwatsubo T. Hasegawa M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), is to that the residue both fibrillization and the conformation of the the and biochemical features of A30P fibrils to WT fibrils formed in the of A30P seeds, the fibrils by electron and protease-resistant core The demonstrated that the features of A30P fibrils to WT fibrils in the of A30P seeds. of structures and features of fibrils has been in other such as proteins and fibrils of protein to protein, the protein formed fibrils fibrils E.M. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). has been with protein and Z. R. Science. 2005; PubMed Scopus Google Scholar, M. P. K. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). have a using epitope-specific antibodies of α-synuclein to between WT fibrils and A30P fibrils analysis revealed the of conformational between α-synuclein and the fibrils WT fibrils by antibodies to the N-terminal region and NAC whereas the by these the N-terminal region and C-terminal region of α-synuclein intramolecular long range interactions (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), is that antibodies to the N terminus and NAC region the in the The that the N-terminal and NAC of α-synuclein are both at the in the fibrils and the NAC region is in the fibrils and the N terminus the NAC region and by the in vitro have that to α-synuclein as three distinct that are by long and structure and that the of is in A30P mutant M. F. I. S. E. F. M. L. B. Biol. PubMed Scopus Google Scholar). is in with by of WT and A30P α-synuclein with epitope-specific fibrillization of the A30P that of WT, the of A30P the fibrillization that of of A30P and the fibrils may explain the in the the and biochemical analyses of seeds, found that fragments are in the of A30P fibrils and that these fragments as seeds. is the after ultracentrifugation is referred to as the in have the of or abnormal in the of is that such or abnormal may to fibrils or in vitro found an of A30P may provide a the of onset in harboring the A30P of nucleation-dependent fibrillization are in WT fibrils formed in the of A30P seeds the and of A30P fibrils whereas A30P fibrils formed in the of WT seeds the character of the WT seeds the features of A30P fibrils analyses of the brains of PD have that α-synuclein from to E. D. P. T. S. A. H. T. O. P. Nat. PubMed Scopus Google Scholar, Y. R.A. C.W. Nat. PubMed Scopus Google Scholar). fibrillization of α-synuclein in the brains of with A30P mutation may in normal We to in the