- Email: [email protected]
Characterisation of isolated α-synuclein filaments from substantia nigra of Parkinson's disease brain
Neuroscience Letters 292 (2000) 128±130
www.elsevier.com/locate/neulet
Characterisation of isolated a-synuclein ®laments from substantia nigra of Parkinson's disease brain R. Anthony Crowther a, Susan E. Daniel b, Michel Goedert a,* a Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK Parkinson's Disease Society Brain Research Centre, Institute of Neurology, University College London, 1 Wake®eld Street, London WC1N 1PJ, UK
b
Received 8 August 2000; received in revised form 10 August 2000; accepted 10 August 2000
Abstract The de®ning neuropathological deposits of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are strongly immunoreactive for a-synuclein. We have shown previously that isolated ®laments from dementia with Lewy bodies and multiple system atrophy brains are labelled in a characteristic fashion by a number of a-synuclein antibodies. Here we have extracted ®laments from substantia nigra of patients with idiopathic Parkinson's disease. Antibodies directed against the carboxy-terminal region of a-synuclein labelled isolated ®laments along their entire lengths. By contrast, an antibody directed against the amino-terminal region of a-synuclein only labelled one ®lament end. These characteristics were identical to those of ®laments extracted from brains of patients with dementia with Lewy bodies and multiple system atrophy. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: a-Synuclein; Lewy bodies; Lewy neurites; Isolated ®laments; Substantia nigra; Parkinson's disease; Dementia with Lewy bodies; Multiple system atrophy
Parkinson's disease (PD) is the second most common neurodegenerative disorder, after Alzheimer's disease. Neuropathologically, it is de®ned by the presence of Lewy bodies and Lewy neurites in the substantia nigra and several other subcortical brain regions [6]. Similar inclusions in the cerebral cortex de®ne dementia with Lewy bodies (DLB), a common cause of dementia. Lewy body pathology is also seen in some cases of Alzheimer's disease, Down's syndrome and neurodegeneration with brain iron accumulation type 1. In all these diseases, Lewy bodies and Lewy neurites are strongly immunoreactive for the presynaptic protein a-synuclein [10,11,14,17]. Moreover, rare familial forms of PD are caused by point mutations in the a-synuclein gene [9,12]. Besides Lewy bodies and Lewy neurites, the glial and neuronal inclusions of multiple system atrophy (MSA) are also immunoreactive for a-synuclein [16±18]. Ultrastructurally, the inclusions of PD, DLB and MSA are made of abnormal ®laments. By immunoelectron microscopy, isolated ®laments from DLB and MSA brains were shown to have similar morphologies and to be decorated in an identical fashion by a number of a-synuclein antibodies * Corresponding author. Tel.: 144-1223-402036; fax: 144-1223402197. E-mail address: [email protected] (M. Goedert).
[2,13,15,16]. This work revealed an unexpected molecular link between DLB and MSA and indicated that a-synuclein may be the major component of the abnormal ®laments. Over the past three years, much work has been devoted to the connection between a-synuclein and PD. By immunoelectron microscopy on tissue sections, Lewy bodies in substantia nigra have been shown to be labelled by a-synuclein antibodies [1]. However, nothing is known about a-synuclein in isolated ®laments from brain stem Lewy bodies. Here, we report the characterisation of a-synuclein ®laments extracted from pooled substantiae nigrae of patients with idiopathic PD. Filaments were extracted from pooled substantiae nigrae of ten neuropathologically con®rmed cases of PD (aged 69± 87 years) by a procedure used previously for extracting ®laments from cingulate cortex of patients with DLB [15] and from frontal cortex and cerebellum of patients with MSA [16]. Procedures for immunoelectron microscopy were as described [4]. The primary antibodies were used at 1:100 (PER1 and PER4) and 1:1 000 (H3C). After reaction with the appropriate secondary gold-conjugated antibody (Sigma Fine Chemicals or BioCell), the grids were stained with 1% lithium phosphotungstate. Micrographs were recorded at a
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0 30 4- 39 40 ( 00) 0 14 40- 3
R.A. Crowther et al. / Neuroscience Letters 292 (2000) 128±130
nominal magni®cation of X40 000 on a Philips model EM208S microscope, as described previously [4]. To identify assembled structures containing a-synuclein, the substantia nigra from the brains of PD patients was extracted using a method that enriches for ®laments that are insoluble in sarcosyl [8]. The resuspended pellets were immunolabelled with a-synuclein antibodies PER1, PER4 and H3C. PER1 was raised against a synthetic peptide corresponding to residues 11±34 of a-synuclein [14], PER4 was raised against recombinant human a-synuclein and recognises the carboxy-terminal half of a-synuclein [15] and H3C was raised against residues 129±143 of zebra ®nch a-synuclein [7] and recognises the last ten amino acids (residues 131±140) of human a-synuclein [13]. The labelled structures corresponded mostly to single ®laments, though occasionally small clumps of ®laments were seen. The morphology was somewhat variable, though all ®laments were straight and unbranched with a length typically between 200 and 600 nm (Fig. 1). The width was variable, with some ®laments about 5 nm wide (Fig. 1C,E,G,H) and others about 10 nm wide (Fig. 1D,I). A few ®laments showed a variation in width from 5 to 10 nm along their length (Fig. 1A,B,F). The ®laments were labelled all along their length by antibodies PER4 (Fig. 1A±C) and H3C (Fig. 1G±I). A quite different pattern was observed with PER1, where only one end of each ®lament was labelled (Fig. 1D±F).
129
The present ®ndings show that isolated, sarcosyl-insoluble ®laments from substantia nigra of PD brain are immunolabelled by a-synuclein antibodies, extending previous light microscopic studies on brain stem Lewy bodies and Lewy neurites [14]. Antiserum PER4 and antibody H3C strongly labelled isolated ®laments along their entire lengths, indicating that they contain a-synuclein as a major component. Labelling by H3C showed the presence of an intact carboxy-terminus of a-synuclein which is exposed on the ®lament surface. By contrast, antibody PER1 only ever labelled one ®lament end, suggesting that the amino-terminal region of a-synuclein is buried in the body of the ®lament and that the ®laments are polar structures. PER1 stained Lewy bodies and Lewy neurites in PD very strongly [14], suggesting the presence of a pool of unassembled a-synuclein in the inclusions of PD. The morphologies of the a-synuclein ®laments seen here from PD brain closely resemble those reported for DLB [15], but not the thicker twisted ®laments seen in MSA [16]. The variable morphology suggests that the 10 nm ®laments may contain 5 nm proto®laments as substructures. The antibody labelling characteristics of the PD ®laments were identical to those described previously for isolated ®laments from DLB and MSA brains [15,16]. Together with the similar morphologies and identical antibody labelling patterns of synthetic a-synuclein ®laments [3,5,13], these ®ndings ®rmly establish that a-synuclein is the
Fig. 1. Decoration of isolated ®laments from substantia nigra of Parkinson's disease brain with a-synuclein antibodies. Filaments were extracted from pooled substantiae nigrae of ten neuropathologically con®rmed cases of idiopathic Parkinson's disease. (A±C) Antiserum PER4, (D±F) antibody PER1, (G±I) monoclonal antibody H3C. The gold particles conjugated to the second antibody appear as black dots. Scale bar, 100 nm.
130
R.A. Crowther et al. / Neuroscience Letters 292 (2000) 128±130
major component of the ®lamentous lesions of PD, DLB and MSA. It follows that a-synuclein is a speci®c and sensitive marker for these pathological inclusions. Dysfunction of a-synuclein and its assembly into ®laments are probably central to the pathobiology of PD, DLB and MSA. However, the mechanisms by which they lead to the degeneration of affected nerve cells and glial cells remain to be discovered. This work was supported by the Medical Research Council and the UK Parkinson's Disease Society. [1] Arima, K., UeÂda, K., Sunohara, N., Hirai, S., Izumiyama, Y., Tonozuka-Uehara, H. and Kawai, M., Immunoelectronmicroscopic demonstration of NACP/a-synuclein-epitopes on the ®lamentous component of Lewy bodies in Parkinson's disease and in dementia with Lewy bodies, Brain Res., 808 (1998) 93±100. [2] Baba, M., Nakajo, S., Tu, P.-H., Tomita, T., Nakaya, K., Lee, V.M.-Y., Trojanowski, J.Q. and Iwatsubo, T., Aggregation of a-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies, Am. J. Pathol., 152 (1998) 879±884. [3] Conway, K.A., Harper, J.D. and Lansbury, P.T., Accelerated in vitro ®bril formation by a mutant a-synuclein linked to early-onset Parkinson's disease, Nat. Med., 4 (1998) 1318± 1320. [4] Crowther, R.A., Straight and paired helical ®laments in Alzheimer's disease have a common structural unit, Proc. Natl. Acad. Sci. USA, 88 (1991) 2288±2292. [5] Crowther, R.A., Jakes, R., Spillantini, M.G. and Goedert, M., Synthetic ®laments assembled from C-terminally truncated a-synuclein, FEBS Lett., 436 (1998) 309±312. [6] Forno, L.S., Neuropathology of Parkinson's disease, J. Neuropathol. Exp. Neurol., 55 (1996) 259±272. [7] George, J.M., Jin, H., Woods, W.S. and Clayton, D.F., Characterization of a novel protein regulated during the critical period for song learning in the zebra ®nch, Neuron, 15 (1995) 361±372. [8] Greenberg, S.G. and Davies, P., A preparation of Alzheimer paired helical ®laments that displays distinct tau proteins by polyacrylamide gel electrophoresis, Proc. Natl. Acad. Sci. USA, 87 (1990) 5827±5831. [9] KruÈger, R., Kuhn, W., MuÈller, T., Woitalla, D., Graeber, M., KoÈsel, S., Przuntek, H., Epplen, J.T., SchoÈls, L. and Riess, O.,
[10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
Ala30Pro mutation in the gene encoding a-synuclein in Parkinson's disease, Nat. Genet., 18 (1998) 106±108. Lippa, C.F., Fujiwara, H., Mann, D.M.A., Giasson, B., Baba, M., Schmidt, M.L., Nee, L.E., O'Connell, B., Pollen, D.A., St. George-Hyslop, P., Ghetti, B., Nochlin, D., Bird, T.D., Cairns, N.J., Lee, V.M.-Y., Iwatsubo, T. and Trojanowski, J.Q., Lewy bodies contain altered a-synuclein in brains of many familial Alzheimer's disease patients with mutations in presenilin and amyloid precursor protein genes, Am. J. Pathol., 153 (1998) 1365±1370. Lippa, C.F., Schmidt, M.L., Lee, V.M.-Y. and Trojanowski, J.Q., Antibodies to a-synuclein detect Lewy bodies in many Down's syndrome brains with Alzheimer's disease, Ann. Neurol., 45 (1999) 353±357. Polymeropoulos, 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. and Nussbaum, R.L., Mutation in the a-synuclein gene identi®ed in families with Parkinson's disease, Science, 276 (1997) 2045±2047. Serpell, L.C., Berriman, J., Jakes, R., Goedert, M. and Crowther, R.A., Fiber diffraction of synthetic a-synuclein ®laments shows amyloid-like cross-b conformation, Proc. Natl. Acad. Sci. USA, 97 (2000) 4897±4902. Spillantini, M.G., Schmidt, M.L., Lee, V.M.-Y., Trojanowski, J.Q., Jakes, R. and Goedert, M., a-Synuclein in Lewy bodies, Nature, 388 (1997) 839±840. Spillantini, M.G., Crowther, R.A., Jakes, R., Hasegawa, M. and Goedert, M., a-Synuclein in ®lamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies, Proc. Natl. Acad. Sci. USA, 95 (1998) 6469± 6473. Spillantini, M.G., Crowther, R.A., Jakes, R., Cairns, N.J., Lantos, P.L. and Goedert, M., Filamentous a-synuclein inclusions link multiple system atrophy with Parkinson's disease and dementia with Lewy bodies, Neurosci. Lett., 251 (1998) 205±208. Tu, P.-H., Galvin, J.E., Baba, M., Giasson, B., Tomita, T., Leight, S., Nakajo, S., Iwatsubo, T., Trojanowski, J.Q. and Lee, V.M.-Y., Glial cytoplasmic inclusions in white matter oligodendrocytes of multiple system atrophy brains contain insoluble a-Synuclein, Ann. Neurol., 44 (1998) 415±422. Wakabayashi, K., Yoshimoto, M., Tsuji, S. and Takahashi, H., a-Synuclein immunoreactivity in glial cytoplasmic inclusions in multiple system atrophy, Neurosci. Lett., 249 (1998) 180±182.
www.elsevier.com/locate/neulet
Characterisation of isolated a-synuclein ®laments from substantia nigra of Parkinson's disease brain R. Anthony Crowther a, Susan E. Daniel b, Michel Goedert a,* a Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK Parkinson's Disease Society Brain Research Centre, Institute of Neurology, University College London, 1 Wake®eld Street, London WC1N 1PJ, UK
b
Received 8 August 2000; received in revised form 10 August 2000; accepted 10 August 2000
Abstract The de®ning neuropathological deposits of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are strongly immunoreactive for a-synuclein. We have shown previously that isolated ®laments from dementia with Lewy bodies and multiple system atrophy brains are labelled in a characteristic fashion by a number of a-synuclein antibodies. Here we have extracted ®laments from substantia nigra of patients with idiopathic Parkinson's disease. Antibodies directed against the carboxy-terminal region of a-synuclein labelled isolated ®laments along their entire lengths. By contrast, an antibody directed against the amino-terminal region of a-synuclein only labelled one ®lament end. These characteristics were identical to those of ®laments extracted from brains of patients with dementia with Lewy bodies and multiple system atrophy. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: a-Synuclein; Lewy bodies; Lewy neurites; Isolated ®laments; Substantia nigra; Parkinson's disease; Dementia with Lewy bodies; Multiple system atrophy
Parkinson's disease (PD) is the second most common neurodegenerative disorder, after Alzheimer's disease. Neuropathologically, it is de®ned by the presence of Lewy bodies and Lewy neurites in the substantia nigra and several other subcortical brain regions [6]. Similar inclusions in the cerebral cortex de®ne dementia with Lewy bodies (DLB), a common cause of dementia. Lewy body pathology is also seen in some cases of Alzheimer's disease, Down's syndrome and neurodegeneration with brain iron accumulation type 1. In all these diseases, Lewy bodies and Lewy neurites are strongly immunoreactive for the presynaptic protein a-synuclein [10,11,14,17]. Moreover, rare familial forms of PD are caused by point mutations in the a-synuclein gene [9,12]. Besides Lewy bodies and Lewy neurites, the glial and neuronal inclusions of multiple system atrophy (MSA) are also immunoreactive for a-synuclein [16±18]. Ultrastructurally, the inclusions of PD, DLB and MSA are made of abnormal ®laments. By immunoelectron microscopy, isolated ®laments from DLB and MSA brains were shown to have similar morphologies and to be decorated in an identical fashion by a number of a-synuclein antibodies * Corresponding author. Tel.: 144-1223-402036; fax: 144-1223402197. E-mail address: [email protected] (M. Goedert).
[2,13,15,16]. This work revealed an unexpected molecular link between DLB and MSA and indicated that a-synuclein may be the major component of the abnormal ®laments. Over the past three years, much work has been devoted to the connection between a-synuclein and PD. By immunoelectron microscopy on tissue sections, Lewy bodies in substantia nigra have been shown to be labelled by a-synuclein antibodies [1]. However, nothing is known about a-synuclein in isolated ®laments from brain stem Lewy bodies. Here, we report the characterisation of a-synuclein ®laments extracted from pooled substantiae nigrae of patients with idiopathic PD. Filaments were extracted from pooled substantiae nigrae of ten neuropathologically con®rmed cases of PD (aged 69± 87 years) by a procedure used previously for extracting ®laments from cingulate cortex of patients with DLB [15] and from frontal cortex and cerebellum of patients with MSA [16]. Procedures for immunoelectron microscopy were as described [4]. The primary antibodies were used at 1:100 (PER1 and PER4) and 1:1 000 (H3C). After reaction with the appropriate secondary gold-conjugated antibody (Sigma Fine Chemicals or BioCell), the grids were stained with 1% lithium phosphotungstate. Micrographs were recorded at a
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0 30 4- 39 40 ( 00) 0 14 40- 3
R.A. Crowther et al. / Neuroscience Letters 292 (2000) 128±130
nominal magni®cation of X40 000 on a Philips model EM208S microscope, as described previously [4]. To identify assembled structures containing a-synuclein, the substantia nigra from the brains of PD patients was extracted using a method that enriches for ®laments that are insoluble in sarcosyl [8]. The resuspended pellets were immunolabelled with a-synuclein antibodies PER1, PER4 and H3C. PER1 was raised against a synthetic peptide corresponding to residues 11±34 of a-synuclein [14], PER4 was raised against recombinant human a-synuclein and recognises the carboxy-terminal half of a-synuclein [15] and H3C was raised against residues 129±143 of zebra ®nch a-synuclein [7] and recognises the last ten amino acids (residues 131±140) of human a-synuclein [13]. The labelled structures corresponded mostly to single ®laments, though occasionally small clumps of ®laments were seen. The morphology was somewhat variable, though all ®laments were straight and unbranched with a length typically between 200 and 600 nm (Fig. 1). The width was variable, with some ®laments about 5 nm wide (Fig. 1C,E,G,H) and others about 10 nm wide (Fig. 1D,I). A few ®laments showed a variation in width from 5 to 10 nm along their length (Fig. 1A,B,F). The ®laments were labelled all along their length by antibodies PER4 (Fig. 1A±C) and H3C (Fig. 1G±I). A quite different pattern was observed with PER1, where only one end of each ®lament was labelled (Fig. 1D±F).
129
The present ®ndings show that isolated, sarcosyl-insoluble ®laments from substantia nigra of PD brain are immunolabelled by a-synuclein antibodies, extending previous light microscopic studies on brain stem Lewy bodies and Lewy neurites [14]. Antiserum PER4 and antibody H3C strongly labelled isolated ®laments along their entire lengths, indicating that they contain a-synuclein as a major component. Labelling by H3C showed the presence of an intact carboxy-terminus of a-synuclein which is exposed on the ®lament surface. By contrast, antibody PER1 only ever labelled one ®lament end, suggesting that the amino-terminal region of a-synuclein is buried in the body of the ®lament and that the ®laments are polar structures. PER1 stained Lewy bodies and Lewy neurites in PD very strongly [14], suggesting the presence of a pool of unassembled a-synuclein in the inclusions of PD. The morphologies of the a-synuclein ®laments seen here from PD brain closely resemble those reported for DLB [15], but not the thicker twisted ®laments seen in MSA [16]. The variable morphology suggests that the 10 nm ®laments may contain 5 nm proto®laments as substructures. The antibody labelling characteristics of the PD ®laments were identical to those described previously for isolated ®laments from DLB and MSA brains [15,16]. Together with the similar morphologies and identical antibody labelling patterns of synthetic a-synuclein ®laments [3,5,13], these ®ndings ®rmly establish that a-synuclein is the
Fig. 1. Decoration of isolated ®laments from substantia nigra of Parkinson's disease brain with a-synuclein antibodies. Filaments were extracted from pooled substantiae nigrae of ten neuropathologically con®rmed cases of idiopathic Parkinson's disease. (A±C) Antiserum PER4, (D±F) antibody PER1, (G±I) monoclonal antibody H3C. The gold particles conjugated to the second antibody appear as black dots. Scale bar, 100 nm.
130
R.A. Crowther et al. / Neuroscience Letters 292 (2000) 128±130
major component of the ®lamentous lesions of PD, DLB and MSA. It follows that a-synuclein is a speci®c and sensitive marker for these pathological inclusions. Dysfunction of a-synuclein and its assembly into ®laments are probably central to the pathobiology of PD, DLB and MSA. However, the mechanisms by which they lead to the degeneration of affected nerve cells and glial cells remain to be discovered. This work was supported by the Medical Research Council and the UK Parkinson's Disease Society. [1] Arima, K., UeÂda, K., Sunohara, N., Hirai, S., Izumiyama, Y., Tonozuka-Uehara, H. and Kawai, M., Immunoelectronmicroscopic demonstration of NACP/a-synuclein-epitopes on the ®lamentous component of Lewy bodies in Parkinson's disease and in dementia with Lewy bodies, Brain Res., 808 (1998) 93±100. [2] Baba, M., Nakajo, S., Tu, P.-H., Tomita, T., Nakaya, K., Lee, V.M.-Y., Trojanowski, J.Q. and Iwatsubo, T., Aggregation of a-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies, Am. J. Pathol., 152 (1998) 879±884. [3] Conway, K.A., Harper, J.D. and Lansbury, P.T., Accelerated in vitro ®bril formation by a mutant a-synuclein linked to early-onset Parkinson's disease, Nat. Med., 4 (1998) 1318± 1320. [4] Crowther, R.A., Straight and paired helical ®laments in Alzheimer's disease have a common structural unit, Proc. Natl. Acad. Sci. USA, 88 (1991) 2288±2292. [5] Crowther, R.A., Jakes, R., Spillantini, M.G. and Goedert, M., Synthetic ®laments assembled from C-terminally truncated a-synuclein, FEBS Lett., 436 (1998) 309±312. [6] Forno, L.S., Neuropathology of Parkinson's disease, J. Neuropathol. Exp. Neurol., 55 (1996) 259±272. [7] George, J.M., Jin, H., Woods, W.S. and Clayton, D.F., Characterization of a novel protein regulated during the critical period for song learning in the zebra ®nch, Neuron, 15 (1995) 361±372. [8] Greenberg, S.G. and Davies, P., A preparation of Alzheimer paired helical ®laments that displays distinct tau proteins by polyacrylamide gel electrophoresis, Proc. Natl. Acad. Sci. USA, 87 (1990) 5827±5831. [9] KruÈger, R., Kuhn, W., MuÈller, T., Woitalla, D., Graeber, M., KoÈsel, S., Przuntek, H., Epplen, J.T., SchoÈls, L. and Riess, O.,
[10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
Ala30Pro mutation in the gene encoding a-synuclein in Parkinson's disease, Nat. Genet., 18 (1998) 106±108. Lippa, C.F., Fujiwara, H., Mann, D.M.A., Giasson, B., Baba, M., Schmidt, M.L., Nee, L.E., O'Connell, B., Pollen, D.A., St. George-Hyslop, P., Ghetti, B., Nochlin, D., Bird, T.D., Cairns, N.J., Lee, V.M.-Y., Iwatsubo, T. and Trojanowski, J.Q., Lewy bodies contain altered a-synuclein in brains of many familial Alzheimer's disease patients with mutations in presenilin and amyloid precursor protein genes, Am. J. Pathol., 153 (1998) 1365±1370. Lippa, C.F., Schmidt, M.L., Lee, V.M.-Y. and Trojanowski, J.Q., Antibodies to a-synuclein detect Lewy bodies in many Down's syndrome brains with Alzheimer's disease, Ann. Neurol., 45 (1999) 353±357. Polymeropoulos, 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. and Nussbaum, R.L., Mutation in the a-synuclein gene identi®ed in families with Parkinson's disease, Science, 276 (1997) 2045±2047. Serpell, L.C., Berriman, J., Jakes, R., Goedert, M. and Crowther, R.A., Fiber diffraction of synthetic a-synuclein ®laments shows amyloid-like cross-b conformation, Proc. Natl. Acad. Sci. USA, 97 (2000) 4897±4902. Spillantini, M.G., Schmidt, M.L., Lee, V.M.-Y., Trojanowski, J.Q., Jakes, R. and Goedert, M., a-Synuclein in Lewy bodies, Nature, 388 (1997) 839±840. Spillantini, M.G., Crowther, R.A., Jakes, R., Hasegawa, M. and Goedert, M., a-Synuclein in ®lamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies, Proc. Natl. Acad. Sci. USA, 95 (1998) 6469± 6473. Spillantini, M.G., Crowther, R.A., Jakes, R., Cairns, N.J., Lantos, P.L. and Goedert, M., Filamentous a-synuclein inclusions link multiple system atrophy with Parkinson's disease and dementia with Lewy bodies, Neurosci. Lett., 251 (1998) 205±208. Tu, P.-H., Galvin, J.E., Baba, M., Giasson, B., Tomita, T., Leight, S., Nakajo, S., Iwatsubo, T., Trojanowski, J.Q. and Lee, V.M.-Y., Glial cytoplasmic inclusions in white matter oligodendrocytes of multiple system atrophy brains contain insoluble a-Synuclein, Ann. Neurol., 44 (1998) 415±422. Wakabayashi, K., Yoshimoto, M., Tsuji, S. and Takahashi, H., a-Synuclein immunoreactivity in glial cytoplasmic inclusions in multiple system atrophy, Neurosci. Lett., 249 (1998) 180±182.