Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of α-Synuclein

α-Synuclein (α-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related α-synulceinopathies. However, whether phosphorylation promotes or inhibits α-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of α-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type α-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated α-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of α-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern α-syn aggregation and toxicity in PD and related α-synulceinopathies. α-Synuclein (α-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related α-synulceinopathies. However, whether phosphorylation promotes or inhibits α-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of α-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type α-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated α-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of α-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern α-syn aggregation and toxicity in PD and related α-synulceinopathies. Mounting evidence from pathologic, genetic, animal model, biochemical, and biophysical studies support the hypothesis that α-synuclein (α-syn) 3The abbreviations used are: α-syn, α-synuclein; PD, Parkinson disease; SEC, size exclusion chromatography; ThT, thioflavin T; TEM, transmission electron microscopy; MTSL, 1-oxy-2, 2, 5, 5-tetramethyl-d-pyrroline-3-methyl)-methanethiosulfonate; CK, casein kinase; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt); MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; LB, Lewy body; WT, wild type; HPLC, high performance liquid chromatography; Bis-Tris, 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)propane-1,3-diol; PBS, phosphate-buffered saline. plays a central role in the pathogenesis of Parkinson disease (PD) and several other neurodegenerative diseases, including Alzheimer disease, multiple system atrophy, dementia with Lewy bodies, Down syndrome, and neurodegeneration with brain iron accumulation, collectively referred to as “synucleinopathies” (1Trojanowski J.Q. Lee V.M. Ann. N. Y. Acad. Sci. 2003; 991: 107-110Crossref PubMed Scopus (130) Google Scholar). Although the exact function of α-syn remains poorly understood, it is thought to play a role in regulating dopamine neurotransmission (2Abeliovich A. Schmitz Y. Farinas I. Choi-Lundberg D. Ho W.H. Castillo P.E. Shinsky N. Verdugo J.M. Armanini M. Ryan A. Hynes M. Phillips H. Sulzer D. Rosenthal A. Neuron. 2000; 25: 239-252Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar), vesicular trafficking (3Cooper A.A. Gitler A.D. Cashikar A. Haynes C.M. Hill K.J. Bhullar B. Liu K. Xu K. Strathearn K.E. Liu F. Cao S. Caldwell K.A. Caldwell G.A. Marsischky G. Kolodner R.D. Labaer J. Rochet J.C. Bonini N.M. Lindquist S. Science. 2006; 313: 324-328Crossref PubMed Scopus (1080) Google Scholar, 4Outeiro T.F. Lindquist S. Science. 2003; 302: 1772-1775Crossref PubMed Scopus (624) Google Scholar), and modulating synaptic function and plasticity (5Kahle P.J. Neumann M. Ozmen L. Haass C. Ann. N. Y. Acad. Sci. 2000; 920: 33-41Crossref PubMed Scopus (83) 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). Increasing evidence suggests that phosphorylation may be an important regulator of α-syn oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity in vivo (7Chen L. Feany M.B. Nat. Neurosci. 2005; 8: 657-663Crossref PubMed Scopus (525) Google Scholar). Immunohistochemical and biochemical studies suggest that the majority of α-syn within LBs from patients with PD and related synucleinopathies (8Fujiwara 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 (159) Google Scholar, 9Anderson J.P. Walker D.E. Goldstein J.M. de Laat R. Banducci K. Caccavello R.J. Barbour R. Huang J. Kling K. Lee M. Diep L. Keim P.S. Shen X. Chataway T. Schlossmacher M.G. Seubert P. Schenk D. Sinha S. Gai W.P. Chilcote T.J. J. Biol. Chem. 2006; 281: 29739-29752Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar, 10Kahle P.J. Neumann M. Ozmen L. Muller V. Jacobsen H. Spooren W. Fuss B. Mallon B. Macklin W.B. Fujiwara H. Hasegawa M. Iwatsubo T. Kretzschmar H.A. Haass C. EMBO Rep. 2002; 3: 583-588Crossref PubMed Scopus (264) Google Scholar, 11Takahashi M. Kanuka H. Fujiwara H. Koyama A. Hasegawa M. Miura M. Iwatsubo T. Neurosci. Lett. 2003; 336: 155-158Crossref PubMed Scopus (116) Google Scholar, 12Hasegawa M. Fujiwara H. Nonaka T. Wakabayashi K. Takahashi H. Lee V.M. Trojanowski J.Q. Mann D. Iwatsubo T. J. Biol. Chem. 2002; 277: 49071-49076Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar) is phosphorylated at Ser-129 (Ser(P)-129). Proteinaceous inclusions formed in cellular and animal models overexpressing WT or mutant α-syn can also be stained with an antibody against Ser(P)-129. A study by Fujiwara et al. (8Fujiwara 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 (159) Google Scholar) reported that in vitro phosphorylated α-syn (at Ser-129, using casein kinase II (CK2)) forms fibrils more readily than unmodified α-syn. Phosphorylation at Ser-129 was also reported to promote the formation of cytoplasmic inclusions in some cell culture models of synucleinopathies (13Smith W.W. Margolis R.L. Li X. Troncoso J.C. Lee M.K. Dawson V.L. Dawson T.M. Iwatsubo T. Ross C.A. J. Neurosci. 2005; 25: 5544-5552Crossref PubMed Scopus (211) Google Scholar). Together, these findings suggested that phosphorylation at Ser-129 plays an important role in modulating α-syn aggregation, LB formation, and toxicity in vivo. However, in vivo studies by Feany and co-worker (7Chen L. Feany M.B. Nat. Neurosci. 2005; 8: 657-663Crossref PubMed Scopus (525) Google Scholar) suggest a lack of correlation between phosphorylation at Ser-129 and the level of α-synfibrillation. Overexpression of the phosphomimic S129D or coexpression of WT α-syn and G protein-coupled receptor kinase 2 (Gprk2), which phosphorylates α-syn specifically at Ser-129, in the Drosophila model of PD results in increased α-syn toxicity without an increase in the number of α-syn inclusions (compared with overexpression of WT α-syn). Interestingly, overexpression of S129A results in a significant increase (4×) in the number of inclusions and suppression of dopaminergic neuronal loss produced by expression of WT human α-syn. We considered that a rigorous examination and comparison of the biochemical and biophysical properties of phosphorylation and and WT α-syn may the as well as the molecular mechanisms by which phosphorylation at Ser-129 may α-syn aggregation and toxicity in vivo. In to the phosphorylation we and the in vitro phosphorylated of α-syn. To better understand the role of the in modulating α-syn aggregation and membrane binding we compared the structural, oligomerization, and membrane binding properties of WT α-syn to those of the phosphorylation mimics (S129E, S129D) as well as the in vitro phosphorylated of α-syn using size exclusion thioflavin and transmission electron results demonstrate that phosphorylation at Ser-129 inhibits than promotes α-syn formation in vitro. important is that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation at on α-syn structure and aggregation properties in vitro. findings have significant implications for in vivo and understanding of the role of α-syn in the pathogenesis of PD and related and of and α-syn using and by used in these studies as J.C. Rochet J.C. 2003; PubMed Scopus Google Scholar). the expression and of and α-syn as R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, W. T. D. G. T.M. V. J. Biol. 2002; PubMed Scopus Google Scholar). To of a a single was into α-syn at for with the mutant was 1-oxy-2, 2, 5, to with α-syn and the was as W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). In Phosphorylation of or mutant α-syn was phosphorylated by at a of was in the of with the and of of α-syn. phosphorylation was at for the and the was with of the was by and the effect of phosphorylation on the aggregation of α-syn, WT α-syn was phosphorylated for at and the was with the to at with for the to the but was not studies the in at a of and formation was by using an as H.A. J. M. R.J. T. J. Biol. 2002; PubMed Scopus Google Scholar). and of from of α-syn was by the of and α-syn in at the the aggregation at the from a α-syn at at for at to was from the and on an system with a and using an was also on and stained with to the of the protein and in and on stained with or to the the membrane and the membrane was with in for at membrane was with the antibody (211) at a of or at a of or α-syn Ser-129 at a of or α-syn at a of at for the membrane was with the antibody from at for was with and with and in a at a of WT or mutant α-syn on at a of with 2 of and stained with 2 of on a electron at with a the phosphorylation and of the phosphorylated of α-syn or was using on a with a and using an or or A was and the was at In the of the HPLC, a was of and and (sodium was in which was by and was with to a of To increase the of formation, of in and at by a membrane to the at and used within and of α-syn in or was with the of to a of of was for 2 at structure of α-syn was by using a of 2 for at in a of α-syn in or and from was from and and from and from of for the A was for the molecular a of in and a in A was on the using a with of was with of and of was on of the and to and with a laser by of the to the that on the of and was with the and to the of α-syn WT or mutant α-syn in at on and was to in the and using F. S. G. J. A. J. 1995; PubMed Scopus Google Scholar) and D. and D. G. of and J. 4: PubMed Scopus Google Scholar). to and using the of for the of mutant and phosphorylated by in the heteronuclear single quantum of the WT protein to the in by and for and to in WT α-syn, phosphorylated α-syn, and S129D α-syn using on WT and mutant α-syn in and as an and V. C.M. PubMed Scopus Google Scholar). as a function of the V. C.M. PubMed Scopus Google Scholar, C.M. J. Scopus Google Scholar). was at was increased from to in a with a of to the of the and to using of α-syn from the of α-syn and and the of C.M. J. Scopus Google Scholar). in from WT and phosphorylated α-syn, from the of between in the and of the for on α-syn on α-syn. or was in in with on a at a of and a of for of and the structural and between and suggest that type of a to phosphorylation at a at in the in a of in to the single by To whether a of Ser-129 by an is to the effect of phosphorylation, we in which Ser-129 was by or To provide insight into the in vivo studies with the phosphorylation the S129A was also and S129A within the human of by and the mutant in and to than as by and To whether of Ser-129 into a is to reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in we the α-syn using We that phosphorylates α-syn more than casein kinase II To phosphorylation at the mutant of α-syn was and phosphorylated in vitro to α-syn To that phosphorylation by at Ser-129, we of the antibody can be as as the of not and increases with and of elucidate the consequences of phosphorylation on the structure and of α-syn, we a of and high in of WT and α-syn, and at the and a of a high of in WT and α-syn in the in of the phosphorylation of WT α-syn by the of and Ser-129 at the in the of the the in the in which of phosphorylated and and the to and α-syn, phosphorylation at was that the of Ser-129 was at its and at its phosphorylated by phosphorylation that the of to by phosphorylation of Ser-129 in α-syn. In addition, for the in α-syn, in with studies H. E. Y. M. T. E. T. Iwatsubo T. Hasegawa M. K. PubMed Scopus Google Scholar). to the of the protein We in WT α-syn and phosphorylated α-syn. to and for WT and phosphorylated not that phosphorylation effect on the structure of α-syn. of WT, and α-syn and phosphorylated WT and α-syn and with a structure and Phosphorylation the of by of the of a the a can be that an of the of a WT α-syn, we a of the of it increased to α-syn, the and α-syn a a of be WT α-syn was its increased by to phosphorylation of α-syn at Ser-129 increased by to of to phosphorylated α-syn increased the to that phosphorylation the of by α-syn to its Phosphorylation the effect of phosphorylation on we of between a specifically and than of of an increase in D. J. Biol. PubMed Scopus Google Scholar). This effect an on the the of between the and the in H. S. 25: PubMed Scopus Google Scholar). the of α-syn was into a to provide an for the the of the the of the the or the of aggregation for α-syn W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). the for WT and phosphorylated α-syn. In WT α-syn, the of a effect to and with in with W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). In phosphorylated α-syn, the was to the of the for and for to To the of phosphorylation at Ser-129 on we in WT and α-syn. of the of WT and α-syn, heteronuclear R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and as R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 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V. 2006; PubMed Scopus Google Scholar), and a of an effect by Ser-129 phosphorylation be on these is to a to binding to To the effect of phosphorylation at Ser-129 on we the structure of the in the and of by In we that phosphorylation does not perturb the of and its to binding to studies to the effect of phosphorylation on membrane binding by multiple in as the the effect of the on the aggregation properties of α-syn, we compared the aggregation of and S129A to that of the WT protein as a function of using the binding and In a the formed more fibrils than WT protein but these not and in other in formation between the and In we that the S129A more and forms more fibrils than the WT and the phosphorylation S129A and α-syn formed fibrils with to that of WT α-syn Phosphorylation the effect of in vitro phosphorylation on α-syn formation, WT α-syn was with in the at for which the to and formation was by and and compared with that to the in the of the We that phosphorylation inhibits α-syn formation, of as from the and studies To the and we also the of α-syn at using by the of the to the that the of α-syn in to phosphorylation remains of formation the of α-syn with with the and In we not of that can be by SEC, we the of in the Phosphorylation at Ser-129 to the of studies that phosphorylates α-syn at and Ser-129 M. J. Koyama A. S. M. Iwatsubo T. L. P.J. Haass C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). To the of phosphorylation at Ser-129 to the of α-syn formation, we the effect of phosphorylation on the of mutant of α-syn. of with results in significant of α-syn to the forms of the of formed significant of the of α-syn 5, A and with the and the of the of the aggregation the of with To demonstrate that of α-syn fibrillogenesis is to phosphorylation at Ser-129, we the phosphorylated of and compared its aggregation properties to that of and WT α-syn. WT, and α-syn to aggregation at with and not fibrils and in a of the and WT α-syn high of as by the increased and the significant in of with and of by However, of in the formation of that from those formed by and WT α-syn the of in a that of α-syn is thought to be phosphorylated at Y. Iwatsubo T. Hasegawa M. Lett. PubMed Scopus Google Scholar), we to whether the forms and of α-syn and the consequences of on the aggregation of α-syn. In the of of we not significant in the of However, in the of we significant of α-syn as by TEM, ThT, and and aggregation of of α-syn was by and of aggregation, of α-syn to level as at findings suggest that of aggregation by is Phosphorylation of α-syn at Ser-129 in the pathogenesis of PD and related However, the exact mechanisms by which phosphorylation the and properties of α-syn in vivo unknown. To understand the structural underlying the effect of phosphorylation on the and properties of α-syn, we the effect of phosphorylation at Ser-129 on the conformational and properties of α-syn. Phosphorylation at Ser-129 in studies that α-syn that by and to and aggregation W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar, K. J. M. C.M. J. Chem. 2005; PubMed Scopus Google Scholar). binding and that aggregation in structure W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). also that high of the of α-syn, to an we the effect of phosphorylation on the of α-syn. by that phosphorylation of α-syn at Ser-129 increased the of α-syn In addition, of and that phosphorylation at Ser-129 phosphorylation the of by α-syn in may its as well as function of α-syn. Ser-129 is the of α-syn that is to its with phosphorylation at these is not to α-syn membrane Phosphorylation at Ser-129 not the binding of α-syn to it with the formation of structure as by the the of the protein in its is also by Ser-129 phosphorylation, with to the of the phosphorylation within the and not into the binding This that of Ser-129 phosphorylation on the function of α-syn, which is to be with its membrane-bound is to be by the of the on the the of Phosphorylation at and used to the structural and consequences of protein phosphorylation, a comparison between the phosphorylation mimics and the phosphorylated of the protein is Our in vitro studies demonstrate that the and S129D do not reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in vitro. of Ser-129 into or not to an of α-syn and the and of to the of that of or to or the effect of phosphorylation on the structure and of α-syn and other Together, these findings the critical of the increased of the to the of and in the structural and consequences of studies have reported that by serine is by In some of these studies it was that the N. B. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), or conformational S. Acad. Sci. S. A. PubMed Scopus Google Scholar) by the than its is for the of can that phosphorylation of α-syn may an important and regulator of α-syn aggregation and with other have important implications for of to elucidate the role of phosphorylation in modulating in vivo and the of results using and for molecular understanding of the consequences of phosphorylation may insight into the of α-syn and the mechanisms by which it to neurodegeneration in PD and related to the effect of phosphorylation on α-syn aggregation and toxicity was by Feany and co-worker (7Chen L. Feany M.B. Nat. Neurosci. 2005; 8: 657-663Crossref PubMed Scopus (525) Google Scholar) in a Drosophila model of PD overexpressing WT, or Interestingly, the aggregation properties of the and and WT α-syn in vivo to we in with WT and the number of S129A forms more (4×) than WT and S129D mutant toxicity and of α-syn expression of the S129A mutant dopaminergic cell loss and increased the of α-syn formed to the WT or S129D for these is that may the formation and of of α-syn than of which may in be However, in vitro studies not with a we that Ser-129 phosphorylation inhibits formation and also the of α-syn to In to modulating the and fibrillogenesis of α-syn, phosphorylation may be in regulating its properties by modulating its with other neuronal synaptic and α-syn with the protein and phosphorylation H. M.S. P. J. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and fibrillogenesis M. M. L. D. Lee J.M. C. P. B. 2005; PubMed Scopus Google Scholar) in vitro and in vivo. binding was to the of α-syn. Phosphorylation at Ser-129, but not was also reported to the of α-syn Hill J. A.A. S. V.L. J. Cell Sci. PubMed Scopus Google Scholar). Together, these findings suggest that phosphorylation within the or may be in regulating the with and other neuronal G. M. K. Lee E. Lee J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R.J. Woods W.S. J.M. J. Biol. PubMed Scopus Google Scholar), N. L. M. N. P. J. B. J. Neurosci. PubMed Google Scholar), (3Cooper A.A. Gitler A.D. Cashikar A. Haynes C.M. Hill K.J. Bhullar B. Liu K. Xu K. Strathearn K.E. Liu F. Cao S. Caldwell K.A. Caldwell G.A. Marsischky G. Kolodner R.D. Labaer J. Rochet J.C. Bonini N.M. Lindquist S. Science. 2006; 313: 324-328Crossref PubMed Scopus (1080) Google Scholar), and Phosphorylation within the or or the of phosphorylation at these also membrane binding and of M. J. Koyama A. S. M. Iwatsubo T. L. P.J. Haass C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R.J. Woods W.S. J.M. J. Biol. PubMed Scopus Google Scholar), an in the of and vesicular Although results demonstrate that α-syn is of binding to in an (compared with the WT we have not the of phosphorylation on membrane which may be Phosphorylation of or LB findings that α-syn is of the of α-syn within of the fibrils formed by is from those formed by the protein the consequences of in structure or not studies to the molecular and cellular that the aggregation of phosphorylated α-syn and whether phosphorylation aggregation or and LB studies have that α-syn with several to α-syn at including K. Lee G. J. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar) and S. M. S. H. M. Koyama S. H. H. T. K. K. M. M. T. S. K. H. C. E. S. T. T. Y. K. T. K. M. I. K. K. Iwatsubo T. M. H. T. J. Neurosci. 2006; PubMed Scopus Google Scholar) in findings with the that the Ser-129 remains in the forms and of α-syn support the hypothesis that phosphorylation of α-syn also within LBs and is not a for α-syn and LB formation in PD. Together, findings suggest that phosphorylation may an important regulator of α-syn aggregation and the of the and in regulating α-syn phosphorylation in for therapeutic strategies for PD and related have as therapeutic for neurodegenerative diseases, including Alzheimer disease M. J. J. 2006; PubMed Scopus Google Scholar), and to be the of of the of and on the development of kinase with