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To whom correspondence should be addressed: Dept. of Neural Plasticity, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585, Japan. Tel.: 81-3-3304-5701 (Ext. 511); Fax: 81-3-3329-8035
* This study was supported by grants from the Ministry of Education, Science and Culture of Japan (to K. A., M. N., and K. U.) and is supported by a fellowship from the Science and Technology Agency of Japan (to M. A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‖ Present address: Dept. of Laboratory Medicine, National Center Hospital for Mental, Nervous and Muscular Disorders, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8551, Japan. ** Present address: Dept. of Demyelinating Disease and Aging, National Institute of Neuroscience, Kodaira, Tokyo 187-8502, Japan.
Increasing evidence suggests that α-synuclein is a common pathogenic molecule in several neurodegenerative diseases, particularly in Parkinson's disease. To understand α-synuclein pathology, we investigated molecules that interact with α-synuclein in human and rat brains and identified tubulin as an α-synuclein binding/associated protein. Tubulin co-localized with α-synuclein in Lewy bodies and other α-synuclein-positive pathological structures. Tubulin initiated and promoted α-synuclein fibril formation under physiological conditions in vitro. These findings suggest that an interaction between tubulin and α-synuclein might accelerate α-synuclein aggregation in diseased brains, leading to the formation of Lewy bodies.
non-Aβ component of Alzheimer's disease amyloid
dementia with Lewy bodies
multiple system atrophy
glial cytoplasmic inclusions
high pressure liquid chromatography
bovine serum albumin
The non-β-amyloid (Aβ)1component of Alzheimer's disease amyloid, or NAC, originally detected in an amyloid-enriched fraction, was shown to be a fragment of its precursor, NACP, by cloning of the full-length cDNA (
). However, the vast majority of cases of neurodegenerative diseases associated with LBs or with α-synuclein pathology, such as Parkinson's disease, dementia with Lewy bodies (DLB), MSA, and the LB variant of Alzheimer's disease, are sporadic, where wild-type α-synuclein has shown to be abnormally accumulated as fibrillar structures. It is therefore likely that at some stage(s) in the fibril formation of α-synuclein, either the nucleation and/or the elongation steps should be somehow accelerated in diseased brains, or alternatively, some degradation process(es) of abnormal structures of α-synuclein might be defective in those patients (
With respect to the amyloidogenesis of Alzheimer's disease, it was demonstrated in vitro that a seed of NAC can accelerate Aβ fibril formation, and conversely, a seed of Aβ can promote NAC fibril formation (
). Similarly, heterogeneous molecules could also be involved in the formation of α-synuclein fibrils, leading to pathological structures of α-synuclein such as LBs.
In this study, we performed a biochemical investigation of molecules that interact with α-synuclein in the human brain, and we identified tubulin as one of the α-synuclein binding/associated proteins. This interaction was confirmed by co-immunoprecipitation experiments. Further, α-synuclein was co-purified with microtubules. Double labeling immunofluorescence revealed that tubulin co-localized with α-synuclein-positive pathological structures such as LBs, Lewy-related neurites in Parkinson's disease and DLB, and glial cytoplasmic inclusions (GCIs) in MSA. In vitro studies demonstrated that α-synuclein fibril formation was initiated and accelerated in physiological media containing a small amount of tubulin.
To our knowledge, this is the first demonstration of the initiation and promotion of α-synuclein fibrillogenesis by an intrinsic neural protein under physiological conditions. We speculate that the interaction between tubulin and α-synuclein may be linked to α-synuclein-associated neurodegenerative diseases.
) was digested with NcoI and BglII, and the DNA fragment was ligated into an expression vector, pET-15b (Novagen), previously linearized with NcoI and BamHI. The resultant pET-α-synuclein was used for transformation ofEscherichia coli BL21(DE3) pLysS, and expression was induced by isopropyl-1-thio-β-d-galactopyranoside. The recombinant α-synuclein was purified using two-dimensional HPLC methods as described previously (
A human brain neocortex (75 g) was homogenized in 9 volumes of 20 mm phosphate buffer, pH 8.0, containing 150 mm NaCl, 1 mmEDTA, 1 μg ml−1 pepstatin, and 10 μg ml−1leupeptin. Insoluble materials were precipitated by centrifugation at 140,000 × g at 4 °C for 60 min. Soluble proteins from three cycles of homogenization and centrifugation were pooled.
Isolation and Characterization of α-Synuclein-binding Proteins from a Human Brain Extract
An affinity column was prepared by the conjugation of cyanogen bromide-activated Sepharose-4B resin (Pharmacia) and recombinant α-synuclein (27 mg) according to the manufacturer's instructions. Soluble proteins from the human brain were loaded onto the column, and the column was then washed with a large volume of loading buffer, 50 mm PBS, pH 7.0, at 4 °C. The adsorbed proteins were eluted with 50 mmsodium citrate, pH 3.0, containing 0.5 m NaCl. An identical control experiment was performed using a glycine-Sepharose-4B column. α-Synuclein-binding proteins were purified from the adsorbed fraction obtained from the α-synuclein affinity column by standard gel-cut methods followed by a reversed phase HPLC and characterized by immunoblotting, mass spectrometry, and microsequencing analyses.
Isolation of Microtubules and Tubulin from Rat Brain
Microtubules were isolated from rat brains by two cycles of a temperature-dependent assembly and disassembly method (
) in PIPES buffer (0.1 m PIPES, pH 6.8, 1 mmEGTA, 1 mm MgCl2) containing 1 mmGTP, 1 μg ml−1 pepstatin, and 10 μg ml−1leupeptin, and tubulin was purified from the microtubule fraction by phosphocellulose (Whatman P11) column chromatography (
). The purity of the tubulin was determined to be >98% by SDS-PAGE and immunoblot analyses. Reassembly of microtubules was performed at 37 °C for 20 min followed by 10 min of incubation with 10 μm taxol for microtubule stabilization.
Immunoprecipitation and Immunoblotting
Primary antibodies against the N-terminal region of α-synuclein (MDV2), the NAC domain (EQV1), and the C-terminal region of α-synuclein (PQE3), and β-synuclein-specific antibody (REE1) were described previously (
). Stringent immunoprecipitation was performed using a fraction of rat brain in radioimmune precipitation buffer (50 mmPBS, pH 7.4, 1 mm EGTA, 1% Nonidet P-40, and 0.5% sodium deoxycholate). Antibodies PQE3, EQV1, and anti-α-tubulin (mouse monoclonal clone DM 1A, Sigma) were added and incubated at 4 °C for 2 h. Antigen-antibody complexes were collected with protein A-Sepharose (Sigma). The beads were washed five times by centrifugation with 1% Triton X-100 in PBS and then boiled for 5 min in SDS-sample buffer. Following SDS-PAGE and immunoblotting, the signals were detected with chemiluminescence reagents (PerkinElmer Life Sciences) and Hyper-ECL film (Amersham Biosciences) as described (
). Anti-β-tubulin (clone TUB 2.1, Sigma), TAU-2 (NeoMarkers), and anti-MAP5 (identical to MAP1b) (Chemicon International) antibodies were also employed. It was confirmed that EQV1 and PQE3 did not cross react with tubulin, whereas tubulin antibodies were unable to react with α-synuclein.
In Vitro Fibrillization
α-Synuclein (100–700 μm) in 25 μl of PBS, pH7.4, was incubated with a fixed amount of tubulin (1 μm) at 37 °C without agitation for 0–15 days with or without 1 μm each MgCl2 and GTP. Control experiments were performed using either incubation with BSA or incubation in the absence of tubulin or α-synuclein. The resulting fibrils were monitored by light scattering at OD 400 nm (
) using 1 μl of the reaction mixture with a Gene Spec III spectrophotometer (Hitachi) and examined by immunoelectron microscopy (immuno-EM).
For immunonegative staining, aliquots of microtubules purified from rat brains or of α-synuclein fibrils in the presence of tubulin were placed on Formvar-coated nickel grids and incubated with one of the following primary antibodies: anti-α-tubulin (1:40), anti-β-tubulin (1:40), TAU-2 (1:25), MDV2 (1:40), EQV1 (1:40), PQE3 (1:40), REE1 (1:40), or anti-Aβ (1:50, DAKO). The immunoreaction was visualized by using 10-nm diameter gold particles (1:20, British BioCell) as described previously (
). For postembedding EM, formalin-fixed tissue from the dorsal motor nucleus of the vagus nerve of a patient with DLB was embedded in LR White resin (London Resin). Ultrathin sections were placed on nickel grids and then incubated with PQE3 and finally with 5-nm diameter immunogold particles (
The co-localization of PQE3- and tubulin-epitopes was studied using formalin-fixed paraffin-embedded sections of the dorsal vagus nucleus of a patient with Parkinson's disease, of cervical sympathetic ganglia of that with DLB, and of the pontine basis of that with MSA. Sections were incubated in a solution of PQE3 (1:400) and either anti-α-tubulin (1:200) or anti-β-tubulin (1:200) and then incubated with FITC-conjugated anti-mouse IgG (1:30, BIOSOURCE Int., Camarillo, CA) and tetraethyl sulforhodamine (Rhodamine) conjugated with anti-rabbit IgG (1:80, BIOSOURCE).
Tubulin Is a Binding/Associated Protein of α-Synuclein
To detect molecules that interact with α-synuclein in the human brain, we subjected human brain proteins to α-synuclein affinity column chromatography. Employing a recombinant α-synuclein column, several proteins were isolated as possible α-synuclein-binding/associated proteins (Fig.1a). After purification of the proteins bound to the α-synuclein column by a reversed phase HPLC, several proteins were determined from their amino acid sequences. One of them with an apparent molecular weight of ∼50 kDa (Fig.1a, arrowhead) was sequenced as MRECISIHVG for the N terminus, and VGINYQPPTVVPGGDLAK for a peptide from theAchromobacter lyticus protease digest. In a data base search (SWISS-PROT), both sequences were found to belong to human α-tubulin. To confirm the interaction between α-synuclein and α-tubulin, immunoprecipitation was carried out, and α-tubulin was co-precipitated with endogenous α-synuclein in the cytoplasmic fraction of the rat brain (Fig. 1b) and vice versa (Fig.1c). β-Tubulin was also co-precipitated using anti-α-synuclein antibodies (not shown), indicating the interaction of α-synuclein with α-β tubulin heterodimers.
α- and β-Synucleins Are Associated with Microtubules
To prove the binding or association of α-synuclein to microtubules, we purified microtubules from rat brains by two cycles of polymerization and depolymerization (
). First, the presence of α-tubulin, β-tubulin, tau, and MAP5 (MAP1b) was confirmed in the purified microtubules by immunoblot analyses (Fig.2a). α-Synuclein was also detected in the same sample with three kinds of anti-α-synuclein antibodies (MDV2, EQV1, and PQE3) that interact with epitopes covering the entire α-synuclein molecule (Fig. 2, b andc). Doublet-like bands with molecular mass of about 19 kDa were detected with MDV2 (anti-α- and β-synuclein), and a band corresponding to the upper one was detected with REE1 (anti-β-synuclein) antibodies (Fig. 2c), indicating that both α- and β-synucleins are present in the purified microtubule fraction. These two bands disappeared when these antibodies were preadsorbed with each of the respective peptide antigens, showing the specificity (Fig. 2c).
The association of α-synuclein with microtubules was then examined by EM. Microtubules with a diameter of 24–26 nm were decorated by gold particles in immuno-EM using either anti-α-tubulin (Fig.3a), or anti-β-tubulin (not shown) antibody. Tau is a well-characterized microtubule-associated protein (MAP). Therefore, TAU-2 was used as a positive control to show its interaction with purified microtubules (Fig. 3b). In contrast to this, anti-Aβ was used as a negative control for this experiment (Fig. 3f). All of the anti-α-synuclein antibodies (MDV2 (Fig. 3c), EQV1 (not shown), PQE3 (Fig.3d), and anti-β-synuclein antibody, REE1 (Fig.3e)) showed positive immunoreactions with purified microtubules. These tubules were not labeled when the primary antibodies for α- and β-synucleins were preadsorbed with the respective antigen (not shown). These results suggest that both α- and β-synucleins are associated with purified microtubules.
Tubulin Stimulates α-Synuclein Fibril Formation
To prove the involvement of tubulin in the fibril formation of α-synuclein, solutions with α-synuclein concentrations ranging from 0 to 700 μm were incubated in the presence or absence of 1 μm tubulin in PBS at 37 °C. Purified tubulin was analyzed by SDS-PAGE (silver stain) and immunoblotting and was confirmed to be free from microtubule-associated proteins, such as tau or MAP5 (MAP1b) (not shown). It is possible that oligomers of tubulin are more effective in acting as seeds for α-synuclein fibrillogenesis. To test this potential, microtubule assembling cofactors, such as MgCl2 and GTP, were added in a separate reaction at minimal concentrations (the commonly used concentrations times 10−3), and the fibril formation was found to be accelerated by these cofactors (Fig. 4,a and b). α-Synuclein was assembled into fibrillar structures only in the presence of 1 μm tubulin as shown by light scattering (Fig. 4, a–c) and by EM (Fig. 4, d–f). Over the course of several hours filaments 10 nm in diameter and about 1 μm in length were formed (Fig. 4d). α-Synuclein fibrils thus produced were examined by immuno-EM with anti-α-synuclein antibodies MDV2 (not shown), EQV1 (Fig. 4e), and PQE3 (not shown), which were previously used to study LBs in Parkinson's disease (
). These filaments were also decorated with anti-α-tubulin (Fig. 4f) and anti-β-tubulin (not shown), but not with negative controls, such as anti-β-synuclein REE1 (not shown) or anti-Aβ (not shown). The dimensions and morphology of these structures are similar to those of α-synuclein filaments in the brain sections of Parkinson's disease (
) (Fig. 4g). α-Synuclein filaments were produced only in the presence of 1 μm tubulin (Fig. 4, a–d) not in solutions containing α-synuclein alone (Fig. 4c; C1) or in other controls (Fig. 4c; C2, C3, andC4). It is notable that the additives alone were not able to stimulate production of α-synuclein fibrils (not shown) but enhanced the fibril production in the presence of tubulin. The morphology of the fibrils produced in the presence of tubulin with additives was identical (not shown) to the morphology of fibrils produced without additives (Fig. 4, d–f). These results indicate that tubulin is able to induce and promote α-synuclein fibril formation under physiological conditions in vitro.
Tubulin Co-localizes with Pathological Structures of α-Synuclein
We subsequently investigated the in vivoco-localization of tubulin and α-synuclein in LBs in the dorsal motor nucleus of the vagus nerve of a patient with Parkinson's disease and cervical sympathetic ganglia of that with DLB, as well as in GCIs of that with MSA using double labeling immunofluorescence methods. The rhodamine-labeled anti-α-synuclein antibody (PQE3) decorated LBs, pale bodies, Lewy-related neurites, and GCIs intensely (Fig.5, a, d, andg). FITC-tagged anti-α-tubulin stained these structures as well (Fig. 5, c, f, and i). These signals overlapped completely or partially when merged (Fig. 5,b, e, and h). β-Tubulin was also detected in these pathological structures by a similar method (not shown).
The discovery of two missense mutations in the α-synuclein gene in certain autosomal dominant familial Parkinson's disease pedigrees (
) suggested a pivotal role of α-synuclein in the onset and progression of Parkinson's disease. This has been substantiated by the following findings. (i) Anti-α-synuclein antibodies detect the major filamentous component of LBs, Lewy-related neurites, and pale bodies (a possible early phase of LB-formation) (
). Because familial Parkinson's disease patients are rare, unknown epigenetic factors may induce fibril formation of wild-type α-synuclein, leading to pathological structures such as LBs. In this study, we searched for potential intrinsic factors that may initiate and/or promote fibril formation of α-synuclein using an α-synuclein column, and we found that tubulin was one of the α-synuclein-binding/associated proteins. Co-localization of tubulin with α-synuclein in LBs and other pathological structures suggested that tubulin might be such a factor.
Employing affinity column chromatography using recombinant α-synuclein and human brain proteins, α-tubulin was identified as an α-synuclein-binding/associated protein. Both the α and β subunits of tubulin were equally co-immunoprecipitated with endogenous α-synuclein in the cytoplasmic fraction of rat brain, which is consistent with a notion that α-tubulin forms a heterodimer with β-tubulin. The interaction between α-synuclein and tubulin was confirmed by copurification of both α- and β-synucleins with microtubules. Immuno-EM studies clearly showed the association of both α- and β-synucleins with purified microtubules as has also been shown for other microtubule-associated proteins such as tau. These results suggest that tubulin may be a physiological binding partner of α-synuclein. However, until the binding affinities are known, it cannot be certain that this interaction between tubulin and α-synuclein is physiologically meaningful. Further studies will be needed to know the physiological role of α-synuclein in association with tubulin.
Because the brain is a rich source of tubulin, we speculate that the interaction between α-synuclein and free tubulin may occur in vivo and that under pathological conditions tubulin might initiate and promote the fibril formation of α-synuclein, leading to pathological structures such as LBs. To test this hypothesis we performed in vitro studies, which showed that α-synuclein fibril formation was in fact initiated and promoted by tubulin. Most importantly, these α-synuclein fibrils were produced only in the presence of a small amount of tubulin under physiological conditions. Immuno-EM studies revealed that the dimensions and morphology of these fibrils made in vitro closely resembled those of the α-synuclein filaments observed in autopsied brain sections of Parkinson's disease and were obviously different from those of microtubules. These results indicate that tubulin is capable of seeding the fibril formation of α-synuclein.
We then investigated the in vivo co-localization of tubulin and α-synuclein in autopsied brains using double labeling immunofluorescence, and we showed that both α-synuclein and α-β tubulin epitopes are co-localized in LBs, pale bodies, Lewy-related neurites, and GCIs in the central and peripheral nervous systems. These results indicate that tubulin co-localizes with pathological structures of α-synuclein in Parkinson's disease, DLB, and MSA.
Tau is a well-characterized MAP, and abnormal aggregates of tau, or tau pathology, are thought to be an early event in the development of neurofibrillary lesions (
). In our in vitroexperiments (Fig. 4) purified tubulin produced α-synuclein fibrils in the absence of tau and MAP5 (MAP1b). More importantly, tau does not co-localize with α-synuclein in LBs in general (
), but tubulin does as shown in Fig. 5, suggesting that the interaction between α-synuclein and tubulin may not be mediated by tau.
The precise mechanism of neurodegeneration in Parkinson's disease brain is unknown. Accumulating evidence suggests that the aggregation of α-synuclein may play a critical role in the pathogenesis of Parkinson's disease (
), although the mechanisms by which α-synuclein is preferentially aggregated in the Parkinson's disease brain remained elusive. As shown here, tubulin is apparently able to initiate the polymerization of α-synuclein, resulting in the formation of α-synuclein fibrils, which may eventually lead to pathological structures such as LBs in diseased brains. Thus, it is possible that some epigenetic elements (e.g. drugs, chemicals, additives in food, or environmental toxins) may affect the assembly/disassembly equilibrium of microtubules. Abnormally increased free tubulin thus produced may trigger α-synuclein fibril formation. If so, those microtubule-disrupting elements can be risk factors for α-synuclein-associated degenerative diseases such as Parkinson's disease, DLB, MSA, and LB variant of Alzheimer's disease. Further investigations of the role of tubulin in the aggregation of α-synuclein may clarify the mechanisms of neurodegeneration in α-synuclein-related diseases.
We thank Drs. Mitsunobu Yoshii, Toshihide Nukada, and Ichiro Sora for valuable comments, Haruko Tonozuka-Uehara for excellent technical assistance, and Yo Shoda and Maho Kato for technical help in photographic work.
Proc. Natl. Acad. Sci. U. S. A.1993; 90: 11282-11286