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J. Biol. Chem., Vol. 275, Issue 28, 21500-21507, July 14, 2000

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Microtubule-associated Protein 1B Is a Component of Cortical Lewy Bodies and Binds -Synuclein Filaments^*

Poul Henning Jensen^§, Khalid Islam^¶, John Kenney, Morten Schallburg Nielsen, John Power^**, and Wei Ping Gai^**

From the  Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark, ^¶ Arpida AG, Munchenstein 4142, Switzerland, the  Institute for Storage Ring Facilities, University of Aarhus, Denmark, and the ^** Department of Human Physiology and Centre for Neuroscience, Flinders University, Aarhus, DK8000C, Bedford Park, SA5042, Australia

Received for publication, January 7, 2000, and in revised form, March 7, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lewy bodies, neuropathological hallmarks of Parkinson's disease and dementia with Lewy bodies, comprise -synuclein filaments and other less defined proteins. Characterization of Lewy body proteins that interact with -synuclein may provide insight into the mechanism of Lewy body formation. Double immunofluorescence labeling and confocal microscopy revealed approximately 80% of cortical Lewy bodies contained microtubule-associated protein 1B (MAP-1B) that overlapped with -synuclein. Lewy bodies were isolated using an immunomagnetic technique from brain tissue of patients dying with dementia with Lewy bodies. Lewy body proteins were resolved by polyacrylamide gel electrophoresis. Immunoblotting confirmed the presence of MAP-1B and -synuclein in purified Lewy bodies. Direct binding studies revealed a high affinity interaction (IC₅₀ ~20 nM) between MAP-1B and -synuclein. The MAP-1B-binding sites were mapped to the last 45 amino acids of the -synuclein C terminus. MAP-1B also bound in vitro assembled -synuclein fibrils. Thus, MAP-1B may be involved in the pathogenesis of Lewy bodies via its interaction with monomeric and fibrillar -synuclein.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Formation of proteinaceous inclusion bodies in the brain is a phenomenon common to several late-onset neurodegenerative diseases (1). These inclusion bodies often contain filamentous components that are dominated by a single protein whose gene may harbor mutations linked to heritable forms of the disease, e.g. tau protein in frontotemporal dementia and parkinsonism linked to chromosome 17, huntingtin in Huntington's disease (1). The dominating protein component normally behaves as a monomeric protein, and factors causing the transition from monomeric to fibrillar states are under intense investigation. Lewy bodies and Lewy neurites, the pathological hallmarks of Parkinson's disease and dementia with Lewy bodies (2), are intraneuronal inclusions comprising -synuclein-containing filaments as the major component (3, 4). Mutations in the -synuclein gene may elicit rare forms of early-onset Parkinson's disease (5, 6).

-Synuclein is a small acidic protein of 140 amino acids. The N-terminal part has 7 imperfect repeats containing the consensus core sequence Lys-Thr-Lys-Glu-Gly-Val. The C-terminal part has no recognized structural elements but has a strong negative charge (7). The precise function of -synuclein is not known, but it can bind various ligands, e.g. brain vesicles and the microtubule-associated protein tau. Through such interactions -synuclein may affect ligand phosphorylation (8), inhibit phospholipase D₂ (9), and act as a negative regulator of dopamine release (10).

-Synuclein is transported from neuronal cell bodies to the presynaptic compartment (11, 12) via several rate components of axonal transport (13). A change in its cellular location occurs in disorders designated as -synucleinopathies, where -synuclein accumulates in cell bodies and proximal neurites as Lewy bodies and Lewy neurites (1). -Synucleinopathies include Parkinson's disease and dementia with Lewy bodies, all featuring similar filamentous neural inclusions. Except for rare forms of familial Parkinson's disease, the vast majority of sporadic Lewy body cases do not have -synuclein gene mutations, indicating that dysfunctions of other gene products may impact on the normal metabolism of -synuclein, so that a similar pathological phenotype is produced. Hence, investigation of -synuclein-interacting components may reveal novel pathogenic pathways.

-Synuclein-containing filaments display a peripheral distribution in brainstem Lewy bodies in Parkinson's disease. This is in contrast to their even distribution in cortical Lewy bodies in dementia with Lewy bodies (3). This difference indicates that cell-specific factors may affect the ordering of the filaments and points to the possible importance of putative -synuclein filament-binding proteins in the process of Lewy body formation. Such proteins might also give information about differences in the neurodegenerative processes of Parkinson's disease and dementia with Lewy bodies, respectively. The microtubule-associated protein 1B (MAP-1B),¹ formerly MAP-5, is a component of brainstem Lewy bodies in Parkinson's disease (14). High levels of MAP-1B are expressed during brain development, whereas in adults its expression is confined to brain regions with significant neuronal morphogenesis e.g. the olfactory bulb (15, 16). The increased level of MAP-1B in brainstem Lewy bodies suggest that the inclusion may perturb MAP-1B metabolism in affected neurons.

Dementia with Lewy bodies is the second most common form of dementia next to Alzheimer's disease (2). Hence, the pathognomonic cortical Lewy bodies are common neuronal lesions, but they have been less intensely studied as compared with brainstem Lewy bodies. Here we have investigated whether MAP-1B is present in cortical Lewy bodies and whether it interacts with -synuclein filaments. For this purpose we adopted a broad experimental approach, including in situ localization in brain tissue, biochemical studies of purified cortical Lewy bodies, and direct binding assays for interactions between MAP-1B and -synuclein monomers or -synuclein filaments.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Miscellaneous

All chemicals were purchased from Sigma and were of analytical grade unless otherwise stated.

Proteins and Peptides

Human full-length -synuclein was expressed in Escherichia coli and purified as described previously (11), except that anion exchange chromatography was performed on a Poros HQ resin at a flow rate of 4 ml/min (Perseptive Biosystems, Allerød, Denmark). The truncated recombinant peptide -synuclein-(55-140) has been previously characterized (17). The peptide -synuclein-(1-95) and peptide -synuclein-(57-94), which was internally truncated for amino acid residues 57-94, were new constructs made by polymerase chain reaction as described previously (17). Peptide -synuclein-(1-95) was purified on a Poros HS cation exchanger (Perseptive Biosystems), whereas peptide -synuclein-(57-94) was purified using a Poros HQ resin. The identities of recombinant peptides were verified by matrix-assisted laser desorption ionization mass spectrometry as described (17). MAP-1B was purified as described (18). Briefly, 4-6-month-old calf brains were homogenized in PIPES buffer and centrifuged at 30,000 × g, 4 °C, for 45 min. The supernatant was collected, adjusted to 1 mM GTP, and incubated at 37 °C for 20 min to promote microtubule polymerization. Polymerized microtubules were removed by pelleting at 30,000 × g, 30 °C, for 45 min. Following further centrifugation for 12 h, 80,000 × g, 4 °C, the supernatant was loaded onto a Mono-Q column pre-equilibrated in MES buffer containing 0.2 M NaCl. Fractions eluted at 0.4 M NaCl were pooled, dialyzed against MES buffer, and loaded on a Mono-S column. MAP-1B (>95% pure) was eluted at 0.33 M NaCl, dialyzed against MES buffer, snap-frozen in liquid nitrogen, and stored at 80 °C until use. The composition of the purified MAP-1B, its phosphorylation status, and microtubule-binding properties have been described in detail (19). Purified MAP-1B was iodinated to a specific activity of 1 Ci/mg using chloramine T as oxidizing agent as described previously for tau protein (8).

Antibodies

Sheep IgG was raised against a synthetic peptide corresponding to a C-terminal portion of human -synuclein (residues 116-131) and affinity purified using the peptide bound to thiopropyl-Sepharose 6B (Amersham Pharmacia Biotech). The specificity for -synuclein was confirmed by absorption of the antibody using the peptide or recombinant human -synuclein (data not shown). This antibody gave strong labeling of Lewy bodies and Lewy neurites in brain sections from Parkinson's disease (20). Antibody AB97/8 was raised against a synthetic peptide corresponding to the C-terminal amino acid residues 116-131 of human -synuclein. Antibody ASY-1 was raised against full-length human recombinant -synuclein. Antibody ASY-3 was raised against a synthetic peptide corresponding to amino acid residues 1-31 of -synuclein. The latter two antibodies were affinity purified using immobilized recombinant -synuclein. The specificity of AB97/8 was confirmed by absorption using corresponding peptides (21). ASY-1 recognized epitopes within the last 45 amino acids as determined by immunoblotting on truncated -synuclein peptides (data not shown). Mouse anti-MAP-1B AA6 was from Sigma. Fluorescein isothiocyanate-donkey anti-sheep, Cy3-donkey anti-mouse, biotin-conjugated donkey anti-sheep IgG, and Cy3-conjugated streptavidin were from Jackson ImmunoResearch Laboratories; horseradish peroxidase-conjugated swine anti-rabbit IgG was from Dakopatt (Glostrup, Denmark), and goat anti-rabbit IgG conjugated to 15 nm diameter gold particles was from Amersham Pharmacia Biotech.

Brain Tissue

Brain tissue used in the present study was obtained from the South Australian Brain Bank. Frontal, temporal, and parietal cortical gray matters were dissected from four cases of pathologically verified dementia with Lewy bodies. Sex and age of death of the cases are as follows: male, 67 years; female, 81 years; male 69 years, and male, 68 years. The brains were removed at autopsy within 24 h after death, bisected at midsagittal plane, with one-half snap-frozen and stored at 70 °C until use. The other half was aldehyde-fixed, blocked, and embedded in paraffin. Neuropathological assessment was conducted on paraffin-embedded sections by a neuropathologist. Numerous Lewy bodies were demonstrated by -synuclein immunostaining in the brainstem, frontal, temporal, and parietal cortices in all four cases. There were no or only occasional neurofibrillary tangles. Control tissues were from three patients dying with non-neurological diseases, with similar age at death and postmortem delay as the patients with dementia with Lewy bodies. There were no significant neuropathologies in the control cases as determined by histological staining (silver, hematoxylin and eosin, and myelin stains) and immunostaining for -amyloid protein, tau, ubiquitin, or -synuclein.

Immunomagnetic Isolation of Lewy Bodies and Lewy Neurites

The procedure was modified from the method for isolating -synuclein-containing glial inclusions from multiple system atrophy brains (22). Centrifugation was carried out with a Beckman J2-MC centrifuge, JA-21 rotor, and Beckman 13.5-ml thick wall polyallomer centrifuge tubes (Beckman Instruments Inc., Palo Alto, CA). Procedures were carried out at 4 °C unless otherwise stated. Cortical gray matter was dissected from the brains, mixed with 4 volumes of homogenization buffer (0.32 M sucrose, 50 mM Tris-HCl at pH 7.4, 5 mM EDTA, leupeptin 1 µg/ml, pepstatin 1 µg/ml, phenylmethanesulfonyl fluoride 17.4 µg/ml), and homogenized in a Dounce homogenizer (Wheaton, NJ), 10 strokes with a loose pestle and 10 strokes with a tight pestle. Sixty milliliters of homogenate (equivalent to 12 g of tissue) were processed in each preparation. The homogenate was filtered through glass wool, diluted 2.5 times with homogenization buffer, and pelleted at 1,000 × g for 10 min. The pellet was washed two times more in homogenization buffer. The pellet of each tube was adjusted to 6 ml with homogenization buffer and Percoll, to a Percoll concentration of 14% (v/v). The sample was overlaid on 2.4 ml of 35% Percoll (v/v in homogenization buffer) and centrifuged at 35,000 × g for 30 min. After centrifugation, myelin material formed a defined band at the top one-third level of the centrifuge tube. Material between the myelin layer and the sample, 35% Percoll interface, was collected and washed three times by centrifugation at 4,000 × g for 10 min in 50 mM Tris-HCl-buffered saline at pH 7.4 containing protease inhibitors. The pellet, approximately 0.5 ml, was suspended in 30 ml of 5 mM MgCl₂, 2 mM EGTA, 10 mM Tris, pH 7.4, containing protease inhibitors, and homogenized at room temperature by 10 strokes in a 50-ml Wheaton glass homogenizer (Wheaton, NJ) fitted with a motor-driven plastic pestle at 250 rpm. The homogenate was filtered through 20-µm nylon mesh (Small Parts Inc., Miami Lakes, FL) and washed three times by 4,000 × g for 10 min in 0.32 M sucrose, 50 mM Tris-HCl, pH 7.4, supplemented with protease inhibitors. The resulting pellet was brought to 24 ml of 12% Percoll (v/v) in the sucrose Tris buffer and overlaid (6 ml each tube) on 2.4 ml of 35% Percoll (v/v in the sucrose Tris buffer). After centrifugation at 35,000 × g for 30 min, the material banding just below the sample, 35% Percoll interface, was collected, approximately 1.5-2 ml from each tube.

The Percoll-fractionated material was pooled, and normal horse serum was added and adjusted to 10% of total volume. Following 30 min of gentle rotation at room temperature, affinity purified sheep anti--synuclein antibody was added at a final concentration of 1.3 µg/ml and incubated for 60 min at room temperature. Following 3 washes in Tris-buffered saline, the sample was resuspended in 10 ml of the same buffer and incubated with biotin-conjugated donkey anti-sheep IgG (1.3 µg/ml) for 30 min at room temperature. Following 3 washes in Tris-buffered saline, the pellet was brought to 3 ml in the same buffer. A sample was taken, labeled with Cy3-conjugated streptavidin (1:800), and the number of Lewy bodies and Lewy neurites counted. The suspension was then adjusted to approximately 10⁶ Lewy bodies and neurites/ml with Tris-buffered saline. Streptavidin-coated magnetic beads (M-280, Dynal, Oslo, Norway) were added, in a ratio of 30 beads per Lewy body or neurite (or 45 µl of magnetic bead suspension per 10⁶ Lewy bodies and neurites). Following incubation for 30 min at room temperature, the magnetic beads were washed 6 times with Tris-buffered saline by using a magnetic particle concentrator (Dynal). The washing process was monitored by taking an aliquot of the sample, labeled with Cy3-conjugated streptavidin, and examined under dark field (all particles) and fluorescence field (Lewy particles and magnetic beads, see "Results"). To elute bound material, the magnetic beads were suspended in solubilization buffer (2% SDS, 8 M urea, 17.4 µg/ml phenylmethanesulfonyl fluoride, 5 mM EDTA in 50 mM Tris-HCl, pH 7.0). The volume of the solubilization buffer was equal to that of original bead suspension used. After overnight incubation at 37 °C, the magnetic beads were removed using the magnetic particle concentrator. The supernatant was subjected to electrophoresis. For every 12 g of brain tissue (starting material), approximately 60-80 µg of total protein was recovered from the dementia with Lewy bodies tissue, whereas less than 6 µg of protein was obtained from normal control brain tissue. To prevent contamination between experiments, each case was processed separately. Cortical gray matter from normal control brains was processed identically as the tissue from patients of dementia with Lewy bodies. Reagents (primary and secondary antibodies, magnetic beads, and SDS/urea sample buffer) were added in amounts equivalent to Lewy body preparations. Control experiments were also conducted with Lewy body preparations including 1) omitting the primary or secondary antibodies and 2) primary antibody preincubated with corresponding peptide (20 µg/ml) before adding to the Lewy body fraction. To monitor the inclusions and non-inclusion contaminants, samples were taken at each step, stained for -synuclein, and examined with light and electron microscopy. For electron microscopy, samples were fixed for 2 h (2% glutaraldehyde, 1% paraformaldehyde, 0.1 M phosphate buffer at pH 7.4), post-fixed for 2 h in 1% OsO₄, dehydrated in cold graded acetone, and embedded in TAAB-embedding resin (TAAB Laboratories, Reading, UK). Ultrathin sections were stained with lead citrate and examined using a JEM 1200 EX electron microscope.

Protein Quantification, Electrophoresis, and Immunoblotting

An elution buffer containing 2% SDS and 8 M urea was used routinely to solubilize Lewy bodies. Total protein contents were determined by using a Bio-Rad DC protein microassay kit on aliquots of solubilized Lewy bodies or equivalent volumes of supernatant from controls. Some Lewy body samples were solubilized with 70% formic acid for 16 h at 37 °C.

For electrophoresis, solubilized inclusion samples were heated at 95 °C for 5 min in 2% SDS, 2% mercaptoethanol loading buffer, and resolved by 6 and 10-20% gradient SDS-polyacrylamide gel electrophoresis for analysis of MAP-1B and -synuclein, respectively. Gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad) or transferred to nitrocellulose membranes. The sample volume for the solubilized inclusions and the human brain cytosol was equivalent to 20 µg of total protein. Recombinant human -synuclein (~200 ng) was used as standard for -synuclein. Rabbit IgGs ASY-1 and ASY-3 to human -synuclein (1:1000) and mouse monoclonal antibody to MAP-1B AA6 (1:1000) were used for immunoblotting. Blots were visualized by chemiluminescence (Amersham Pharmacia Biotech).

Binding Assays

Microplate Assay-- The ¹²⁵I-MAP-1B binding to immobilized -synuclein was performed essentially as described previously for ¹²⁵I-tau binding (8) but using Polysorb microtiter plates (NUNC, Kamstrup, Denmark) and a coating concentration of 50 µg/ml recombinant -synuclein.

Fibril Binding Assay-- The term fibril was used here for -synuclein filaments formed in vitro to distinguish from the -synuclein-containing filaments extracted from Lewy body disease brain tissue. All procedures were performed at 4 °C unless otherwise stated. Recombinant human -synuclein was solubilized in fibril buffer (30 mM MOPS, pH 7.4, 0.02% NaN₃) and concentrated to 20 mg/ml in a Centriprep device with 10-kDa cut off (Amicon Inc. Bedford, MA). Prior to initiation of filament formation, the -synuclein solution was centrifuged for 20 min at 500,000 × g in a TLA 120.1 rotor in an Optima TLX centrifuge (Beckman Instruments) to remove aggregated material, and the supernatant was used for fibril formation. -Synuclein fibrils were formed by incubating the supernatant at 37 °C for 7 days. The fibril solution was then diluted with 9 volumes of phosphate-buffered saline containing 0.1% bovine serum albumin. Ligand binding was performed by incubating -synuclein fibril solution with 900 pM ¹²⁵I-labeled MAP-1B or ¹²⁵I-labeled transferrin (Sigma) for 30 min at 37 °C. Transferrin was chosen as a negative control as it is a soluble protein with no reported relation to Lewy bodies. Bound and free tracers were separated by placing 80 µl of the incubate over 120 µl of 40% sucrose cushion and centrifugation in an Airfuge (Beckman Instruments) at maximal speed for 20 min. One hundred and fifty microliters of the supernatant were collected, and the remaining supernatant was carefully removed from the tube using a cotton swab. The tip of the tube containing the pellet was cut and quantified by -counting (Packard Instrument Co.).

Immunohistochemical Light and Electron Microscopy and Immunoelectron Microscopy

For light microscopy, tissue blocks were dissected from various cortical or brainstem regions from fresh-frozen brains, embedded in Tissue-Tek (Sakura Fine Technical Co., Ltd., Tokyo, Japan), sectioned (10 µm) with a cryostat microtome, and thawed onto gelatin-coated glass slides. Sections were air-dried, blocked with 20% normal horse serum for 1 h, and then incubated with sheep anti--synuclein (2.6 µg/ml) and mouse anti-MAP-1B AA6 (1:300) overnight. Following three washes, sections were incubated with fluorescein isothiocyanate-donkey anti-sheep (1:200) and Cy3-donkey anti-mouse (1:200) and examined with conventional or confocal fluorescence microscopy.

Immunoelectron microscopy was used to confirm that fibrous structures prepared as above were indeed composed of -synuclein and not the artifact of preparations. Three different experiments were performed as follows: 1) labeling with C-terminal directed ASY-1 and gold-conjugated anti-rabbit IgG; 2) preimmune rabbit IgG- and gold-labeled second antibody; and 3) gold-labeled second antibody alone. The last two experiments were controls for the specificity of ASY-1 staining of -synuclein fibrils. All antibodies were used at 70 µg/ml and diluted in fibril buffer (30 mM MOPS, pH 7.4, 0.02% NaN₃). -Synuclein fibril preparations (1 mg/ml) were pipetted onto carbon-coated copper grids (3 µl for each grid) and allowed to stand for 1 min. The grids were blocked with 0.1% goat serum for 10 min and incubated with primary antibody or control antibody for 1 h. The grids were then washed twice for 10 min with fibril buffer, blocked for 10 min with 0.1% goat serum, and incubated with gold-conjugated secondary antibody for 1 h. After washing for 10 min with 0.1% goat serum and 10 min with fibril buffer, the grids were stained with 1% aqueous uranyl acetate for 1 min. The grids were air-dried and examined in a Philips 208 electron microscope. Photomicrographs were taken at × 12,500, 25,000, or 31,500.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Immunofluorescence double staining for -synuclein and MAP-1B was used to investigate the presence of MAP-1B in cortical Lewy bodies. In routine formaldehyde-fixed pathological sections, only a small proportion of -synuclein-positive cortical Lewy bodies (<5%) and none of Lewy neurites were MAP-1B-positive (not shown). In freshly prepared sections without formaldehyde treatment, however, most -synuclein-positive Lewy bodies (Fig. 1, A-C) and Lewy neurites (Fig. 1, D-F) were MAP-1B-positive. The MAP-1B staining of Lewy bodies and neurites was abolished when the AA6 antibody was preabsorbed with purified bovine MAP-1B (not shown). A quantitative analysis conducted in sections from three case of dementia with Lewy body indicated that more than 80% of cortical Lewy bodies (of total 130 Lewy bodies counted) and neurites (of total 320 Lewy neurites counted) contained MAP-1B immunoreactivity. Moreover, confocal laser scanning microscopy revealed a high degree of spatial overlapping between the two proteins in both Lewy bodies and Lewy neurites (Fig. 1, A and D versus B and E). Despite concentrated MAP-1B staining in Lewy bodies and Lewy neurites, the cytoplasm of Lewy body-containing neurons did not show increased MAP-1B immunoreactivity as compared with adjacent neurons that did not contain Lewy bodies or neurons from normal cases.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Colocalization of -synuclein and MAP-1B in cortical Lewy bodies and neurites. Confocal laser scanning microscopy of frozen sections prepared from frontal cortical brain tissue of dementia with Lewy bodies cases were incubated with (i) sheep anti--synuclein IgG and labeled with fluorescein isothiocyanate-conjugated anti-sheep antibodies (green in A and D), and (ii) mouse monoclonal anti-MAP-1B and Cy3-conjugated anti-mouse antibodies (red in B and E). C and F show merged images of the double-stained Lewy body (A and B) and neurite (D and E), respectively, and regions of overlapping between -synuclein and MAP-1B immunoreactivities are yellow. Scale bars are shown in A and D.

Isolated Lewy bodies were used to investigate whether MAP-1B is specifically associated with Lewy bodies, and the biochemical nature of MAP-1B and -synuclein extracted from Lewy bodies. For this purpose, we modified a method that was previously used to isolate glial inclusions from multiple system atrophy brain tissue (22). Lewy bodies were first enriched through multiple density gradient centrifugation and then captured from crude Lewy body fractions by sequential binding of 1) sheep antibodies against -synuclein, 2) biotin-conjugated donkey anti-sheep IgG, and 3) streptavidin-coated magnetic beads. This procedure permits constant monitoring of the enrichment and purity of inclusions during isolation. Fig. 2 (A-C) shows a purified Lewy body preparation with streptavidin-coated magnetic beads forming numerous aggregates with -synuclein-positive Lewy bodies and neurites. These aggregates were not present in normal control brain preparations nor were they seen in Lewy body preparations if the anti--synuclein antibodies were omitted or preabsorbed with -synuclein antigen (not shown, see Ref. 22). The structure of purified Lewy bodies and Lewy neurites was better appreciated by confocal scanning laser microscopy (Fig. 2C). Transmission electron microscopy revealed the filamentous nature of the inclusions (Fig. 2D).

View larger version (87K): [in this window] [in a new window]   Fig. 2.   Purification of Lewy bodies from brain tissue of dementia with Lewy bodies. A-C are fluorescent microphotographs showing the final preparation of Lewy bodies with attached streptavidin-coated magnetic beads. The preparation was initially labeled by sheep anti--synuclein IgG and subsequently visualized by Cy3-conjugated streptavidin, which bound to excess biotinylated secondary antibodies in Lewy bodies and neurites, and on the magnetic beads, thus rendering all particles of the preparation visible. A, low magnification of the preparation showing magnetic bead-bound aggregates. The arrow points to a solitary magnetic bead for comparison. B, higher magnification of the preparation showing a Lewy body (asterisk) surrounded by many magnetic beads (arrow) and Lewy neurites (double arrow) associated with magnetic beads. C, confocal microscopic image of the preparation showing the relationship between magnetic beads (one is pointed by an arrow) and Lewy bodies (one is marked by an asterisk) and Lewy neurites (double arrow). D, electron micrograph showing a Lewy body, consisting of filamentous, granular, and vesicular structures, surrounded by magnetic beads (MB).

Purified Lewy bodies and Lewy neurites were solubilized and resolved by reducing SDS-urea gel electrophoresis and analyzed by protein staining and immunoblotting. Coomassie Blue staining of gels revealed a smeary pattern containing multiple protein bands (data not shown). In human brain cytosol fractions, MAP-1B antibody AA6 detected a high molecular weight band, with some reactivity residing at the interphase of the stacking gel and the separating gel (Fig. 3, lane 5). In solubilized inclusions, multiple bands with molecular sizes ranging from 130 to 220 kDa were detected (Fig. 3, lane 6). These bands were abolished when the antibodies were preabsorbed with purified bovine MAP-1B (Fig. 3, lane 7). There was no apparent aggregated MAP-1B reactivity at the top of separation gel, as compared with prominent -synuclein aggregates (Fig. 3, lanes 6 versus 2, see description following), suggesting the MAP-1B peptides may not be covalently linked to -synuclein aggregates.

View larger version (94K): [in this window] [in a new window]   Fig. 3.   MAP-1B and -synuclein immunoreactive peptides in human brain cytosol and extracts of isolated Lewy bodies. Approximately 20 µg of total protein of SDS/urea extracts of isolated Lewy bodies (lanes 2-4, 6 and 7) and the cytosol fraction from a normal control brain (lanes 1 and 5) were resolved by 6 (lanes 5-7) and 10-20% gradient (lanes 1-4)-reducing SDS-polyacrylamide gel electrophoresis. Lane 4 was the 70% formic acid extract of purified Lewy bodies. Lanes 1 and 2 were probed with C-terminally directed anti--synuclein AB97/8; lanes 3 and 4 by N-terminally directed anti--synuclein ASY-3; and lanes 5 and 6 by anti-MAP-1B antibody AA6. The AA6 antibody used in lane 7 was preabsorbed with purified bovine MAP-1B. The top bands in lanes 2, 3, and 5 corresponded to the border between the stacking gel and the resolving gel. Lane 4 was from a separate experiment. Molecular size markers (in kDa) are shown on the left of the blots.

In human brain cytosol fractions, the -synuclein C-terminally specific antibody AB97/8 detected a single band of approximately 20 kDa, as expected for -synuclein monomer (Fig. 3, lane 1). In solubilized inclusions, however, the antibody detected a small amount of monomeric -synuclein and multiple bands that were probably -synuclein dimers and oligomers (Fig. 3, lane 2). There was considerable reactivity at the top of the separating gel and appeared as a high molecular weight smear, indicative of -synuclein aggregates (Fig. 3, lane 2). The same staining pattern was also detected in solubilized Lewy bodies by the -synuclein N-terminally specific antibody ASY-3 (Fig. 3, lane 3). In urea-SDS-solubilized Lewy bodies, no apparent -synuclein fragments of molecular mass less than 20 kDa were detected by either AB97/8 or ASY-3 (Fig. 3, lanes 2 and 3). When Lewy bodies were solubilized with 70% formic acid, in addition to prominent monomeric and polymer -synuclein bands, a band of approximately 15 kDa was also detected by the ASY-3 antibody (Fig. 3, lane 4), indicative of a C-terminally truncated -synuclein peptide. The intensity of the 15-kDa band was approximately 1/5th that of the monomeric -synuclein band as estimated by the exposure time (data not shown).

The presence of MAP-1B and -synuclein in isolated Lewy bodies and the spatial overlap of the two proteins in Lewy bodies in situ suggest possible interactions between -synuclein and MAP-1B. We examined this possibility by using a solid phase binding assay, in which ¹²⁵I-labeled bovine MAP-1B was used as tracer to bind immobilized recombinant human -synuclein. Purified MAP-1B consisted of the MAP-1B heavy chain as the dominant species and a minor contaminant of 80 kDa, as demonstrated by Coomassie Blue staining of polyacrylamide gels (Fig. 4, inset, lane 1). Upon iodination, the labeled MAP-1B tracer comprised essentially the MAP-1B heavy chain as demonstrated by autoradiography (Fig. 4, inset, lane 2). The binding assay demonstrated that ¹²⁵I-labeled MAP-1B specifically bound to immobilized recombinant human -synuclein (Fig. 4), and time course experiments indicated the binding reached a plateau between 7 and 9 h at 4 °C (data not shown). Therefore, all subsequent solid phase binding experiments were performed with a 16-h incubation. As shown in Fig. 4, the interaction between MAP-1B and -synuclein was of high affinity, with a half-saturation concentration of approximately 20 nM (IC₅₀ = 22 nM ± 6 nM; mean ±1 S.D. from three separate experiments). The MAP-1B tracer bound immobilized -synuclein to the same extent as -synuclein, whereas the binding to -synuclein only was 40% of the binding to -synuclein (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Direct binding analysis of 125I-MAP-1B to -synuclein. Recombinant -synuclein, immobilized in microtiter plates, was incubated with 50 pM 125I-MAP-1B and increasing concentrations of unlabeled MAP-1B (). The ordinate represents the percentage of bound/free (B/F) ligand, and the abscissa represents the concentration of free ligand. The value represents the mean ± 1 S.D. of four replicates in 1 of 3 independent experiments. Inset, lane 1, purified bovine MAP-1B (5 µg) were mixed with 10,000 cpm 125I-MAP-1B, resolved by 8-16% reducing SDS-polyacrylamide gel, and stained by Coomassie Blue. Lane 2, autoradiogram of the same gel. The molecular size markers (in kDa) are shown on the left.

The MAP-1B-binding sites were identified by using immobilized -synuclein peptides with different truncations, including -synuclein-(55-140) with an N-terminal deletion of amino acid residues 1-54, -synuclein-(57-94) with an internal truncation of residues 57-94, and -synuclein-(1-95) lacking the C-terminal residues 96-140 (Fig. 5, inset). The purity of these peptides was confirmed by Coomassie Blue staining of polyacrylamide gels. The unusually slow electrophoretic migration of -synuclein-(55-140) peptide is due to its large negative charge, a phenomenon that has been described (17). As shown in Fig. 5, truncation of the -synuclein C-terminal 45 residues caused 75% reduction in MAP-1B binding, whereas truncations in other -synuclein regions had no significant effect. The reduced binding to -synuclein-(1-95) peptide was not due to inefficient immobilization of the peptide to microplates, because the microplates retained their binding capacity for ¹²⁵I-A, a ligand whose binding sites have been mapped to the N-terminal portion of -synuclein (17, 23). Thus, MAP-1B is a high affinity ligand whose binding sites are in the C-terminal one-third of -synuclein.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Localization of MAP-1B-binding sites in -synuclein. Recombinant human full-length -synuclein-(1-140) and truncated -synuclein peptides were immobilized on microtiter plates and incubated with 50 pM 125I-MAP-1B in the presence or absence of 500 nM unlabeled MAP-1B. Specific binding was defined as the binding in the absence of unlabeled MAP-1B minus that in the presence of unlabeled MAP-1B. The ordinate shows the specific binding to -synuclein peptides divided by the binding to -synuclein-(1-140), expressed as mean ± 1 S.D. of four replicates in 1 of 2 independent experiments. Inset, Coomassie Blue staining of the recombinant -synuclein peptides (3 µg each lane) that were used for the binding assay experiments. From lanes 1-4, -synuclein-(1-140), -synuclein-(55-140), -synuclein-(57-94), and -synuclein-(1-95). The molecular size markers (in kDa) are shown on the left.

Antibody mapping studies suggest that the C-terminal segment of -synuclein, either in filaments extracted from Lewy body disease brains or in fibrils formed in vitro, is exposed on the surface of filaments (24-27). We suspect that MAP-1B, which binds the -synuclein C-terminal portion, is capable of binding in vitro formed -synuclein fibrils. We designed an -synuclein fibril binding assay that was based on the cosedimentation of -synuclein fibrils and bound tracer through a 40% sucrose cushion during ultracentrifugation. Fibrils were first formed in vitro and then incubated with tracers. After ultracentrifugation the material recovered from the pellets consisted of straight and curved fibrils (Fig. 6, upper panel). Immunogold electron microscopy confirmed they were indeed -synuclein-containing fibrils. These fibrils were labeled by the primary antibody ASY-1 whose epitopes have been localized within the last 45 amino acids of -synuclein (Fig. 6, A and B). Replacing ASY-1 with a preimmune IgG resulted in no labeling of the fibrils (Fig. 6, C). The diameters of the fibrils were around 8 nm, in agreement with the size previously reported for -synuclein fibrils (25-27).

View larger version (40K): [in this window] [in a new window]   Fig. 6.   125I-MAP-1B bind to in vitro-formed -synuclein fibrils. -Synuclein fibrils were formed by incubating recombinant human -synuclein at 37 °C for 7 days. Ligand binding was measured by incubating -synuclein fibrils (2 mg/ml) with 900 pM 125I-labeled MAP-1B or 125I-labeled transferrin. Bound and free tracer was then separated by centrifugation through a 40% sucrose cushion, and the fibril-containing pellet was analyzed by immunoelectron microscopy or gamma-counting. Upper panel shows -synuclein fibrils recovered from the pellet, following labeling with antibody ASY-1 and secondary antibodies coupled to 15-nm gold particles. A, straight and slightly curved fibrils. A fibril apparently consists of two protofibrils (indicated by arrow) that are approximately 8 nm in diameter. B, dense meshes of -synuclein fibrils. C, negative control, processed as in A and B, but with the antibody ASY-1 replaced by preimmune rabbit IgG. The bar in C is 100 nm and also applies to A and B. Lower panel, tracer binding to the pelleted material after incubation with monomeric (M) and fibrillar (F) -synuclein. 125I-Transferrin, which does not bind -synuclein, was used as negative control. The ordinate represents the percentage of the tracer recovered from the pellet. The bar represents the mean ± 1 S.D. of three independent experiments. The coefficient of variation between experiments was 0.15.

The fibril binding assay indicated that MAP-1B indeed bound -synuclein fibrils, as determined by cosedimentation of ¹²⁵I-MAP-1B tracer with preformed -synuclein fibrils (Fig. 6, lower panel, middle column). The tracer recovered from the pellet was not due to the MAP-1B that bound to the small amount of -synuclein monomers present in the fibril preparations (see below), as ¹²⁵I-MAP-1B that was incubated with -synuclein monomers could not be detected by our fibril binding assay (Fig. 6, lower panel, left column). The binding of MAP-1B to -synuclein fibrils was specific, because the negative control, ¹²⁵I-labeled transferrin, was not cosedimented after incubation with -synuclein fibrils (Fig. 6, lower panel, right column). The preparation of fibrous -synuclein, incubated with the ¹²⁵I-MAP-1B tracer, did contain some non-aggregated -synuclein as shown by Coomassie Blue staining of polyacrylamide gels of the non-pelleted material (data not shown). These -synuclein monomers/oligomers might have competed with fibrous -synuclein for ¹²⁵I-MAP-1B binding. Thus, we consider the fibril binding assay, although useful for identifying ligands capable of binding of -synuclein fibrils, is not suitable for quantitative analysis of the concentration of ligand-binding sites on the fibrils or the binding affinity.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lewy bodies are chemically complex structures. Numerous proteins have been localized in Lewy bodies mostly by immunohistochemistry (28), and one could argue that MAP-1B is just one among many of these proteins. One limitation with immunohistochemistry is that it cannot determine how well a given protein is integrated into the Lewy body (28). To determine whether MAP-1B is an integrated component of Lewy bodies or merely trapped in the inclusions, we immunoisolated cortical Lewy bodies from brain tissue of patients dying with dementia with Lewy bodies. Despite vigorous washings and multiple centrifugation, MAP-1B is present in isolated Lewy bodies as demonstrated by immunofluorescence microscopy and Western blotting, suggesting that MAP-1B is tightly associated with Lewy bodies.

The presence of MAP-1B in both brainstem (14) and cortical Lewy bodies suggests MAP-1B may play a general role in the pathogenesis of Lewy bodies. The distinct morphological differences between brainstem and cortical Lewy bodies may thus depend on cell-specific factors other than MAP-1B. The ability of MAP-1B to bind both monomeric and filamentous -synuclein suggests the possibility that MAP-1B may be involved in all stages of Lewy body formation, from initial conversion of -synuclein from monomers to filaments to continued recruitment of -synuclein molecules to already formed Lewy bodies.

The high level of MAP-1B in Lewy bodies is remarkable since in the adult brain, MAP-1B expression is restricted to neurons that are subjected to high levels of neuronal plasticity (15, 16). In contrast, widespread MAP-1B expression occurs during development. At least two possible alternatives may explain the high level of MAP-1B in Lewy bodies; either Lewy bodies may stimulate the expression of MAP-1B, or the inclusions may merely sequester MAP-1B from its normal catabolism. We favor the latter as ubiquitin conjugates are abundant in Lewy bodies, and the 130-220-kDa MAP-1B immunoreactive peptides in the purified Lewy bodies may thus reflect inefficient Lewy body-associated in vivo proteolysis. An alternative explanation for the multiple MAP-1B fragments associated with Lewy bodies could be that they represent disease-associated translations of previously reported alternative MAP-1B transcripts (29, 30). This would be analogous to the expression of certain tau isoforms in specific neurodegenerative diseases (1). This question may be answered by immunoblotting using antibodies raised against epitopes specific to certain isoforms of the MAP-1B molecule, since attempts to isolate such MAP-1B cDNAs from postmortem brain material is less likely to succeed.

We find that -synuclein aggregates from Lewy bodies are difficult to solubilize to monomeric -synuclein by 8 M urea, 2% SDS, indicative of their tightly packed filamentous nature. The urea-SDS treatment did not release any truncated -synuclein peptides from Lewy bodies. Following extensive formic acid extraction, however, a minor band at 15 kDa representing a C-terminally truncated -synuclein peptide is detected by the -synuclein N-terminally specific antibody ASY-3. This suggests that the C-terminally truncated -synuclein peptide is tightly associated with -synuclein filaments. In a previous study, Baba and colleagues (4) have detected a similar C-terminally truncated peptide as the major -synuclein immunoreactivity from isolated Lewy bodies. This discrepancy may be due to the use of different antibodies for immunoblots or because the procedures used in Baba et al. (4) and our study isolate different populations of Lewy bodies due to e.g. different binding epitopes, -synuclein in the present study versus polyubiquitin chains. Sian et al. have also isolated Lewy bodies using ubiquitin antibodies and magnetic beads (36). Otherwise the procedure of Baba et al. (4) may leave some proteases non-inhibited or may have used brain tissue that have been subjected to a longer postmortem delay.

We have shown that MAP-1B binds the C-terminal segment of monomeric -synuclein with high affinity, suggesting the -synuclein-MAP-1B interaction could play a role in normal function rather than restricting it to Lewy body biology. Such a role could involve regulation of MAP-1B post-translational modifications that are known to differ according to developmental stages and compartmental localizations of MAP-1B in neurons (for review see Ref. 31). This is supported by our recent demonstration that -synuclein binds microtubule-associated protein tau to facilitate kinase-catalyzed phosphorylation of tau (8). Immunoelectron microscopic studies suggest that the C-terminal segment of -synuclein is exposed in both Lewy body filaments and in vitro formed -synuclein fibrils. Considering the tight packaging of -synuclein molecules in filaments, one would expect that the high local concentration of the -synuclein C-terminals would lead to accumulation of C-terminally directed ligands, e.g. MAP-1B and tau (8). The later prediction is supported by the demonstration of tau proteins in some Lewy bodies (32).

Previous studies of -synuclein fibrils formed in vitro have focused on the morphology, antigenic properties, and the effects on fibril formation of Parkinson's disease-causing mutations and C-terminal truncations (25-27, 33, 34). In the present study we have used for the first time -synuclein fibrils in a functional assay to demonstrate their ligand-binding properties. The -synuclein fibril binding assay is based on the classical microtubule binding assays. It will be instrumental for discriminating between ligands for monomeric and fibrillar -synuclein, as no significant monomer-associated tracer is pelleted, and the amount of pelleted tracer displayed a clear dose response as a function of fibril concentration (data not shown). Furthermore, a specificity in the fibril binding is demonstrated by the lack of pelleted ¹²⁵I-transferrin, which does not bind -synuclein as determined by the solid phase binding assay. However, our investigation did not fulfill the definition of specificity as a saturable binding. In preliminary experiments addition of 100-fold excess of unlabeled MAP-1B failed to decrease the pelleted tracer. This might be due to the high concentration of ligand-binding sites or the presence of non-pelletable monomeric binding sites. Nevertheless, the assay may be useful for identifying novel ligands for -synuclein fibrils by performing preparative ligand binding experiments in relevant biological extracts using the fibrils as bait. We find the fibril-binding assay to be a valuable new technique for qualitatively demonstrating ligand binding to Lewy body-like -synuclein fibrils, whose structural composition may be manipulated by the Parkinson's disease mutations or deletions of specific peptide sequences.

The recent demonstration of overexpression of human -synuclein in mice leading to loss of dopaminergic functions and formation of both cytoplasmic and intranuclear inclusions will greatly facilitate the understanding of the role -synuclein may play in neurodegeneration (35). The morphological difference between intranuclear inclusions in the transgenic mice and Lewy bodies in human patients and the absence of -synuclein filaments in the inclusions in mice suggests other factors may be involved in the formation of Lewy bodies (35). The copurification of MAP-1B and -synuclein peptides with Lewy bodies suggests that isolated Lewy bodies and preformed synthetic -synuclein fibrils could be useful for identifying other unknown proteins or protein modifications potentially involved in Lewy body diseases.

	ACKNOWLEDGEMENTS

We thank Drs. Colin Masters and Vladimir Buchmann for the antibody 97/8 and recombinant human -synuclein, respectively. We thank Helen Saibil and Arvid Maunsbach for use of electron microscopic facilities. We also thank Lis Hygom for excellent technical assistance.

	FOOTNOTES

^* This work was supported by the Danish Parkinson Foundation, Danish Medical Research Council Grant 9802803, the Aarhus University Research Foundation, the National Health and Medical Research Council of Australia, the National Health and Medical Research Council Brain Bank Consortium, and the Australian Research Council.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.

^§ To whom correspondence should be addressed: Dept. of Medical Biochemistry, Bldg. 170, University of Aarhus, DK-8000 Aarhus C, Denmark. Tel.: 4589422856; Fax: 4586131160; E-mail: phj@biokemi.au.dk.

Published, JBC Papers in Press, April 10, 2000, DOI 10.1074/jbc.M000099200

	ABBREVIATIONS

The abbreviations used are: MAP-1B, microtubule-associated protein 1B; PIPES, 1,4-piperazinediethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

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