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J. Biol. Chem., Vol. 280, Issue 7, 5703-5715, February 18, 2005

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p25 Stimulates -Synuclein Aggregation and Is Co-localized with Aggregated -Synuclein in -Synucleinopathies^*

Evo Lindersson, Ditte Lundvig, Christine Petersen, Peder Madsen, Jens R. Nyengaard, Peter Højrup¶, Torben Moos||, Daniel Otzen**, Wei-Ping Gai, Peter C. Blumbergs, and Poul Henning Jensen¶¶

From the Institute of Medical Biochemistry and Stereological Research and Electron Microscopy Laboratory, University of Aarhus, Aarhus, DK-8000, Denmark, the ^¶Department of Molecular Biology, University of Southern Denmark, Odense, DK-5000, Denmark, the ^||Department of Medical Anatomy, The Panum Institute, University of Copenhagen, Copenhagen, DK-2200, Denmark, the ^**Department of Life Sciences, Aalborg University, Aalborg, DK-9220, Denmark, the Department of Human Physiology, Flinders University School of Medicine, Adelaide, SA 5042, Australia, and the Department of Neuropathology, Institute of Medical and Veterinary Science, Adelaide, SA 5042, Australia

Received for publication, September 10, 2004 , and in revised form, December 6, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Aggregation of the nerve cell protein -synuclein is a characteristic of the common neurodegenerative -synucleinopathies like Parkinson's disease and Lewy body dementia, and it plays a direct pathogenic role as demonstrated by early onset diseases caused by mis-sense mutations and multiplication of the -synuclein gene. We investigated the existence of -synuclein pro-aggregatory brain proteins whose dysregulation may contribute to disease progression, and we identified the brain-specific p25 as a candidate that preferentially binds to -synuclein in its aggregated state. Functionally, purified recombinant human p25 strongly stimulates the aggregation of -synuclein in vitro as demonstrated by thioflavin-T fluorescence and quantitative electron microscopy. p25 is normally only expressed in oligodendrocytes in contrast to -synuclein, which is normally only expressed in neurons. This expression pattern is changed in -synucleinopathies. In multiple systems atrophy, degenerating oligodendrocytes displayed accumulation of p25 and dystopically expressed -synuclein in the glial cytoplasmic inclusions. In Parkinson's disease and Lewy body dementia, p25 was detectable in the neuronal Lewy body inclusions along with -synuclein. The localization in -synuclein-containing inclusions was verified biochemically by immunological detection in Lewy body inclusions purified from Lewy body dementia tissue and glial cytoplasmic inclusions purified from tissue from multiple systems atrophy. We suggest that p25 plays a pro-aggregatory role in the common neurodegenerative disorders hall-marked by -synuclein aggregates.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The group of -synucleinopathies is dominated by the frequent neurodegenerative disorders Parkinson's disease (PD),¹ Lewy body dementia (LBD), a Lewy body variant of Alzheimer's disease, and multiple system atrophy (MSA) (1–4). Their unifying hallmark is the development of aggregates of the 140-amino acid -synuclein (AS) protein, which is deposited in intracellular inclusions. The inclusions comprise the Lewy body-type of inclusions in the neuronal cell body, the Lewy neurites in axons, and the oligodendrocytic glial cytoplasmic inclusions in MSA. AS can play an active role in the degenerative processes as evidenced by studies of families with autosomal dominant early onset PD and LBD caused by missense mutations in the AS gene (5–7) and the overexpression of the wild type protein caused by gene multiplications of the AS locus (8–10). However, the role of AS in the sporadic diseases is less clear. The frequency of AS aggregation in human diseases has given rise to extensive studies of the process in vitro. These studies have revealed that this aggregation represents a nucleation-dependent process (11). The transition from monomeric AS to filamentous aggregates is characterized by a lag phase during which a build-up of soluble nucleation-competent oligomeric AS species takes places. The rapid filament growth does not occur until these structures have reached a critical concentration. This process is stimulated by several factors like the pathogenic missense mutations A30P and A53T (12–15), increased concentration of AS (16), decreased pH (16), elevated temperature (16), proteolytic truncations of the acidic C terminus (17, 18), molecular crowding (19), phosphorylation of Ser-129 (20), and oxidative modifications like nitrations (21) and dopamine conjugation (22). The reverse inhibitory effects on AS aggregation have also been demonstrated, because the aggregation-incompetent synucleins, -synuclein and -synuclein, may block the process (23, 24).

The mechanisms governing AS aggregation in the sporadic -synucleinopathies remain unexplained, as is the case for tau and A in sporadic Alzheimer's disease, but they are likely to involve perturbations in age-dependent, genetic, and environmental balances of pro- and anti-aggregative factors. We identified p25 as an AS filament-binding protein, which in substoichiometric amounts stimulates AS aggregation. p25 was originally co-purified with a tau kinase preparation from bovine brain (25) as a protein localized to oligodendrocytes (26). Functionally, p25 is subject to phosphorylation by several kinases (25, 27, 28), and it acts as a microtubule-associated protein that causes the formation of aberrant bundles of microtubules (29). In the -synucleinopathies, PD, LBD, and MSA, the cellular expression of p25 is abnormal in the degenerating oligodendrocytes and neurons where it co-localizes with AS in Lewy bodies, Lewy neurites, and glial cytoplasmic inclusions. This suggests that by bringing the pro-aggregative p25 into contact with AS, abnormal p25 expression in neurons and AS in oligodendrocytes may actively contribute to the degenerative process. This would seem to represent a novel prodegenerative factor in the group of -synucleinopathies.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Miscellaneous and Proteins—Recombinant human full-length AS-(1–140), AS-(1–125), AS-(1–110), AS-(1–95), and -synuclein were expressed in Escherichia coli and purified as described previously (30–32). This procedure was followed by an additional reverse phase-high pressure liquid chromatography purification step on a Jupiter C18 column (Phenomenex, Torrance, CA) in 0.1% trifluoroacetic acid with an acetonitrile gradient. The proteins were subsequently aliquoted, lyophilized, and stored at –80 °C. The synthetic peptide AS-(109–140) was originally described by Nielsen et al. (32). The affinity-purified sheep anti--synuclein antibody has been described previously (33). The rabbit FILA-1 IgG was raised against aggregated AS as described previously (34). The anti-bovine p25 peptide antibody originally described in Ref. 25 was kindly provided by Dr. Miho Takahashi, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.

Recombinant Human p25—The human p25 cDNA was amplified by reverse transcription-PCR from a human fetal brain mRNA library (Clontech) using the following primers, p25 5', 5'-CACCCATGGCTGACAAGGCCAA-3', and p25 3', 5'-CACGGATCCCTACTTGCCCCCTTGCAC-3'. For the expression of a hexahistidine-p25 fusion protein, the PCR fragment was inserted into the pT7-PI vector (35). After sequencing of the vector, the protein was expressed and purified on a Talon metal affinity resin (Clontech) according to the manufacturer's recommendations. For expression of native p25, the PCR fragment was inserted into the pET-11d vector (Novagen, San Diego) and sequenced. For protein purification, E. coli BL21 (DE3) cells (Stratagene, La Jolla, CA) were transformed, pelleted, and lysed by sonication on ice in buffer A (50 mM NaH₂PO₄, pH 8.2). The soluble proteins were heated to 100 °C for 10 min whereupon the heat-denatured proteins were removed by centrifugation. Heat-stable proteins were loaded on a Poros HS50 cation column (PerSeptive Biosystems, Foster City, CA), which was eluted by a double linear gradient first into buffer B (1 M NaCl, 50 mM NaH₂PO₄, pH 8.2) and subsequently into buffer C (1 M NaCl, 50 mM NaH₂PO₄, pH 12). p25 eluted early after the pH was raised above pH 8.2. The final purification and buffer exchange were performed on a GF-75 gel filtration column (Amersham Biosciences) that had been pre-equilibrated with 120 mM NaCl, 20 mM sodium phosphate, pH 7.4 (PBS), supplemented with1 mM dithioerythritol. Dithioerythritol was included to avoid unspecific dimerization via the three free Cys residues. The purified p25 was >95% pure as determined by densitometric scanning of Coomassie Blue-stained gels. The identity of human p25 was ascertained by 15 cycles of Edman degradation on a P1000A protein sequencer (Agilent, Palo Alto, CA) of the intact protein with perfect agreement to residues 2–16 of the published sequence (SwissProt accession number O94811 [GenBank] ).

Biophysical Characterization—Urea denaturation experiments were carried out at p25 concentrations of 2.5 µM in 2 mM dithioerythritol, PBS, pH 7.4, at 25 °C. Fresh 10 M urea stock solutions were prepared on a daily basis. All CD studies were performed on a Jasco J-715 spectropolarimeter (Jasco Spectroscopic Co., Tokyo, Japan) with a Jasco PTC-348W temperature control unit. Spectra were recorded in a 0.1-cm path length cuvette with resolution at 0.2 nm, bandwidth at 1.0 nm, sensitivity at 50 millidegree, response at 2.0 s, and speed of 20 nm/min at 25 °C. Three scans were averaged to yield the final spectrum. Protein concentrations were 20 (far-UV CD, 250–205 nm) and 200 µM (near-UV CD, 320–250 nm).

p25-1 Antibody Production—Rabbits were immunized with a fusion protein of a hexahistidine tag linked to the N terminus of p25, and serum was affinity-purified on p25 immobilized to CNBr-activated Sepharose (Amersham Biosciences) followed by protein A chromatography. The resulting rabbit IgG p25-1 was dialyzed against 1 mM EDTA, PBS, pH 7.4, and stored at –20 °C.

Iodination of p25—Purified recombinant human p25 (6 µg) was iodinated essentially as described previously for tau (36), yielding a tracer with a specific activity of 4.8 x 10⁵ Ci/mol.

-Synuclein Aggregate Analyses—Aggregates of recombinant AS-(1–140), AS-(1–125), AS-(1–110), AS-(1–95), and A-(1–40) were formed as described for AS-(1–140), AS-(1–95), and A-(1–40) and quantified by Coomassie Blue-stained SDS-PAGE after their isolation from the pellet following density gradient centrifugation (34). Aggregate-binding proteins, isolated after aggregate co-sedimentation, were identified after nonequilibrium pH-gradient gel electrophoresis (NEPHGE) for the resolution of basic protein (37) followed by mass spectrometric tryptic peptide mapping of individual protein spots (34).

Photoaffinity Cross-linking of -Synuclein Aggregate-bound Proteins—A chemical cross-linking assay was established in order to investigate protein-protein interactions involving aggregated AS. For this purpose, we used the cross-linker sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) (Pierce). SASD is a heterobifunctional cross-linking reagent that contains a photoreactive cross-linking group and an amine-reactive group and produces a 18.9-Å bridge between conjugated molecules (38). The bridge of SASD contains a disulfide bridge that provides cleavability after conjugation. Its photosensitive part can be radiolabeled with ¹²⁵I prior to the conjugation reaction. After cleavage by a reducing agent, the radioactive label will remain attached to the protein conjugated by photoactivation and thus allow identification of the labeled protein. SASD radiolabeling was performed by using the oxidizing agent, IODO-GEN (1,3,4,6-tetrachloro-3,6-diphenylglycouracil, Pierce), which was plated on the surface of a glass tube prior to iodination. The procedures for radiolabeling and cross-linking were as follows. 20 µg of IODO-GEN was dissolved in 100 µl of chloroform and added to a glass tube. The chloroform was slowly evaporated under N₂. SASD was dissolved in dimethyl sulfoxide to 50 mM and further diluted in PBS, pH 7.4, to a final concentration of 0.5 mM. Next, 200 µl of the SASD solution and 100 µCi of Na¹²⁵I were added to the IODO-GEN-coated tube and incubated for 30 s at room temperature. Removing the SASD solution from the IODO-GEN-coated tube and supplementing it with 20 mM KI to quench any residual iodinating activity terminated the iodination reaction. The ¹²⁵I-SASD was subsequently incubated with the purified AS aggregates (200 µg) in PBS, pH 7.4, for 1 h at room temperature to create the ¹²⁵I-SASD-protein complex. The SASD-labeled aggregates were then placed on a 40% sucrose cushion and centrifuged at 15,000 rpm for 30 min to remove free ¹²⁵I and ¹²⁵I-SASD, which stayed in the supernatant. The ¹²⁵I-SASD-AS aggregate tracer, with a specific activity of 1 x 10⁵ Ci/mol AS monomer in the aggregate, was resuspended from the pellet in PBS and used immediately. The SASD-modified AS aggregate tracer was then incubated with p25 for 30 min at 37 °C during continuous shaking. All the above procedures were carried out in the dark, and all reaction vessels were covered by aluminum foil. After incubation, the reaction mixtures were exposed to UV irradiation (Desega Uvis lamp, Copenhagen, Denmark) at 254 nm and at 10 cm from the source for 10 min at room temperature to activate the photoreactive cross-linker. The disulfide bridge in SASD was cleaved by 2% (v/v) -mercaptoethanol (Applichem, Darmstadt, Germany), and samples were analyzed by SDS-PAGE and autoradiography.

¹²⁵I-p25 Solid Phase Binding Assay to -Synuclein-(1–140) Aggregates—Aggregates of recombinant AS (25 µg/ml) in 200 mM NaHCO₃, pH 9.6, were sonicated and immobilized on Polysorp microtiter plates (Nunc, Copenhagen, Denmark) for 2 h on ice, and residual protein-binding sites were blocked by incubation with 5% bovine serum albumin (Sigma) for another 2 h. After rinsing, the wells were incubated with 50 pM ¹²⁵I-p25 in the presence of various concentrations of unlabeled competitor peptides in binding buffer (150 mM NaCl, 2 mM MgCl₂, 1 mM EGTA, 0.01% bovine serum albumin, 20 mM Hepes, pH 7.4) for 16 h at 4 °C. Unbound ligand was removed by rinsing the wells three times with 250 µl of binding buffer, and bound tracer was quantified by -counting (Packard Cobra II, Albertville, MN) after release with 250 µl of 10% SDS.

Determination of -Synuclein/p25 Aggregate Formation—Monomeric AS (340 µM) was incubated in the absence and presence of p25 (3.4 and 10.2 µM) in 0.1% -mercaptoethanol, 120 mM NaCl, 50 mM Tris, pH 7.4, at 37 °C. After 3 days, insoluble material of the 25-µl sample was isolated by density gradient centrifugation. The pellets were resuspended in 30 µl of 8 M urea, 4% SDS overnight at 37 °C, and supplemented with 30 µl of dithioerythritol-containing SDS-loading buffer. The samples were subjected to SDS-PAGE and analyzed by Coomassie staining.

Thioflavin T Fluorescence Assay—Samples were prepared as above, and samples were taken at different time points. Fluorescence measurements were performed at final concentrations of 10 µM protein and 20 µM thioflavin T in 90 mM glycine-NaOH, pH 8.5, using a Wallac Victor³ 1420 (PerkinElmer Life Sciences) multilabel counter (excitation at 450 nm, emission at 486 nm) with 1-s integration.

Brain Tissue—Samples of substantia nigra from sporadic PD (n = 4), LBD (n = 3), and control subjects (n = 4) were obtained from the Netherlands Brain Bank and the National Health and Medical Research Council South Australian Brain Bank. Samples of the cerebral cortex, temporal cortex, and basal ganglia were obtained from MSA cases (n = 3) from the NHMRC South Australian Brain Bank. The brain samples from this bank were bisected, and one-half was fixed with 4% formaldehyde and 2% picric acid, and the other was sliced, fresh frozen, and stored at –80 °C. Control samples were obtained among patients without previous neurological disease before death and no pathological changes in sections of the substantia nigra pars compacta. In the human autopsies, the PD diagnosis was based on loss of neuromelanin pigment and an excessive number of Lewy bodies in the remaining substantia nigra pars compacta neurons in eosin-stained sections as described previously in detail (see Refs. 39 and 40). To verify the distribution of p25 in optimally treated brain tissue, material from four brains was collected from adult male Wistar rats processed for immunohistochemistry (40).

Immunohistochemistry of Paraffin Sections—Paraffin sections (6 µm) were obtained from pathologically confirmed PD (n = 4), LBD (n = 4), MSA (n = 4), and age-matched control cases (n = 3) without significant brain pathology. The sections were deparaffinized, treated with antigen retrieval (boiling in 1 mM EDTA, pH 8.0, for 10 min), and placed for 10 min in 3% H₂O₂ to bleach the endogenous peroxidase reactivity. The sections were blocked with 20% normal horse serum for 60 min and then incubated overnight at 4 °C with affinity-purified rabbit p25-1 antibody (1:1000) and biotinylated donkey anti-rabbit IgG (1:200, The Jackson Laboratory) for 2 h. Following incubation with streptavidin-biotin-peroxidase complex for 1 h, the sections were developed using 3,3-diaminobenzidine tetrahydrochloride as chromogen. Control staining was done by omitting primary antibodies. The rat brains were cut at 40 µm and reacted by floating freely. All sections were incubated overnight at 4 °C with polyclonal rabbit anti-p25-1 antibody diluted 1:200 or sheep anti-human -synuclein antibody (Abcam, Cambridge, UK) diluted 1:200. Specific binding was verified by using either biotinylated swine anti-rabbit antibody diluted 1:500 (Dakopatts, Copenhagen, Denmark) or biotinylated rabbit anti-sheep antibody diluted 1:500 (Abcam, Cambridge, UK), followed by amplification with biotinylated tyramide using TSA Indirect antibody (PerkinElmer Life Sciences) diluted 1:100 for 5 min followed by horseradish peroxidase-conjugated streptavidin-biotin complex for 30 min. The sections were developed as described for the human specimens.

Immunofluorescence Double Staining of Paraffin Sections—To achieve double labeling, human brain sections were pretreated as described above and simultaneously incubated overnight with affinity-purified sheep anti-AS antibody (1:300) in combination with affinity-purified rabbit p25-1 antibody (1:200), and subsequently for 2 h in fluorescent dye-tagged secondary antibodies. Secondary antibodies were Cy3-conjugated anti-sheep IgG and Cy2 conjugated anti-rabbit IgG (1:100, The Jackson Laboratory). Control staining was performed by omitting the primary antibodies. Following the completion of fluorescence immunostaining, sections were mounted on gelatin-coated slides, and buffered glycerol was used as coverslips. Sections were examined by using a BioRad confocal laser-scanning microscope and software package (MRC 1024, Bio-Rad). Lewy bodies were identified in relevant brain regions of PD or LBD cases as neuronal inclusions immunopositive for AS. A Lewy body (usually <30 µm) was scanned along the z axis (through the thickness of the section) at 2-µm increments to determine whether it was completely represented within the thickness of the section (50 µm) and its equatorial plane. This plane was defined at the z axis coordinate where it exhibited the clearest central halo or largest circumference. Images were captured at x2400 magnification.

Immunolabeling of Isolated Lewy Bodies—Lewy body and Lewy neurites were isolated essentially as described previously (33, 41). The pellet, enriched in Lewy bodies and Lewy neurites along with other cellular components, was smeared on slides and air-dried. Following a 10-min fixation with 4% formaldehyde and 2% picric acid in phosphate buffer, pH 7.4, the smears were immunostained for AS (sheep anti-AS, 1:300) and p25 antibody (rabbit p25-1, 1:400). Following labeling with Cy3-conjugated anti-sheep IgG and Cy2-conjugated anti-rabbit IgG (1:100, The Jackson Laboratory, West Grove, PA), the slides were placed on a coverslip using buffered glycerol and were examined by using a Bio-Rad confocal laser-scanning microscope as described above. Control staining was done by omitting the primary antibodies.

Immunoelectron Microscopy—Immunoelectron microscopic localization of recombinant p25 on AS fibrils was performed essentially as described previously for the demonstration of the binding of 20 S proteasomes to AS fibrils (34). In brief, 0.6 µg of recombinant p25 was incubated with 35 µg of AS filaments resuspended at a total volume of 20 µl for 16 h at 4 °C, whereupon the filaments with associated p25 were isolated by density gradient centrifugation. These filaments were resuspended in 70 µl of distilled water and applied onto carbon-coated nickel grids. The primary antibodies used were rabbit p25-1 IgG and nonimmune rabbit IgG, both at 0.1 mg/ml.

Quantitative Electron Microscopic Determination of -Synuclein Aggregate Formation—Quantitative electron microscopy was used to determine whether p25 stimulated the formation of AS filaments or other kinds of aggregates, e.g. amorphous aggregates, and to quantify the profilamentous effect. Monomeric AS (300 µM) was incubated alone and with 3 µM p25 in 120 mM NaCl, 50 mM Tris, pH 7.4, at a total volume of 200 µl at 37 °C with five independent samples from each situation. Insoluble material from 25 µl of each sample was isolated by density gradient centrifugation after 3 days of incubation, and the pellets were resuspended in 50 µl of H₂O, whereupon 3 µl of the resuspended aggregates were pipetted onto carbon-coated nickel grids and subjected to negative staining (34). The ultrastructure of negatively stained grids was examined with a Philips CM10 electron microscope (Eindhoven, the Netherlands) using the AnalySIS software version 3.1 (Soft Imaging System, Münster, Germany). All measurements were performed blindly at a magnification of x3600. Each grid was examined in a meandering fashion using systematic, uniformly random sampling where the step length was 1700 µminthe x direction and 85 µminthe y direction. We used point counting with a 14 x 9 grid and area per test point equal to 46.3 µm² to estimate the area fraction of the fibers as shown om Equation 1,

(Eq. 1)

where P(fiber) is the test point hitting the AS fibers and P(grid) the test point hitting the grid. This method assumes that the fibers are round in order to avoid the effect of overprojection.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Identification of p25 as an -Synuclein Filament-binding Protein—Cellular proteins binding to aggregated AS may represent targets or modifiers of the toxic effect of the aggregates (34) or modulators of the aggregative process. We used in vitro formed AS aggregates to search for such proteins in rat brain cytosol, deploying an AS aggregate sedimentation technique (34). A range of proteins co-sedimented with the AS aggregates upon cytosol incubation with the AS aggregates, and two-dimensional NEPHGE was used to resolve basic proteins. Fig. 1A (right panel) presents a part of a silver-stained gel that demonstrates a strong protein spot with a weaker, slightly more acidic isoform that was only present when the cytosol was incubated with the AS aggregates. This protein was absent in the cytosol control (Fig. 1A, left panel) and the AS aggregate control (data not shown). This spot was subjected to mass spectrometric peptide mapping (Fig. 1C), and the spectrum identified the protein as the rat homologue of human p25 with the identified peptides covering 33% of the entire protein (Table I). Rat p25 identification was further confirmed by immunoblotting with the rabbit anti-p25 peptide IgG (Fig. 1D), which was raised against a peptide in bovine p25 (25). The AS aggregate-binding property was not restricted to rat p25, as incubation of a human brain extract with AS aggregates enabled the co-sedimentation of human p25 (Fig. 1B). p25 is a brain phosphoprotein (see Ref. 25; data not shown) and rat brain cytosol comprises multiple pI isoforms of p25 as compared with the single immunoreactive spot for recombinant human hexahistidine-p25 (Fig. 1D, upper versus lower panel). All these isoforms could associate with AS aggregates as demonstrated by co-sedimentation (Fig. 1D, middle panel).

View larger version (32K): [in this window] [in a new window]   FIG. 1. Identification of p25 as an -synuclein filament-binding protein. A, rat brain cytosol was incubated in the absence and presence of 35 µg of -synuclein (AS) aggregates, whereupon the samples were subjected to density gradient centrifugation. Pellets were resolved by nonequilibrium pH-gradient two-dimensional gel electrophoresis and subjected to silver staining. Electrophoresis resolved the basic proteins in the pH range 7–11, which left the acidic AS out of the gel. Left panel, part of the gel containing the cytosolic sample with some insoluble proteins stained at the bottom. Right panel, similar part of the gel containing AS aggregate + cytosol sample. Arrow indicates AS aggregate-associated protein subjected to mass spectrometric peptide mapping. Arrowhead marks putative, more acidic isoform of the same protein. Anode and cathode localizations are presented on top of the gels. The fractionation corresponding to molecular weight (MW) is displayed to the left. B, immunoblot demonstration of the binding of human brain p25 to AS aggregates. Human brain detergent extract was co-incubated with AS aggregates as in A. Pellets were subjected to reducing SDS-PAGE and immunoblotting using rabbit anti-p25 antibody p25-1. Lane 1, 10% of inputs of brain extract. Lane 2, pellet from extract without aggregates. Lane 3, pellet from extract plus AS aggregates containing associated human brain proteins. Molecular size markers in kDa are indicated to the left. C, mass spectrum of tryptic digests of protein spot indicated by arrow in A. The gel spot was reduced and alkylated with iodoacetamide prior to digestion. The abscissa shows the mass divided by charge (m/z) of individual ions, and the ordinate shows the signal intensity corresponding to the ions. The peptide pattern was searched against the NCBInr protein data base using the Mascot search program (Matrix Science, UK) and showed a significant correlation with rat p25 (peak assignment in Table I). p25 was identified in three independent experiments using different rats. D, rat brain cytosol and AS aggregate-binding proteins from cytosol were isolated by centrifugation, and the pellets were resolved by two-dimensional gel electrophoretic analysis as in A, but subjected to immunoblotting with the p25-1 antibody. Input demonstrates multiple pI isoforms in cytosol. The sample to be incubated with the aggregates was supplemented with 50 ng of hexahistidine-tagged recombinant human p25 fusion protein. The lower part demonstrates immunoreactivity of unmodified recombinant hexahistidine-tagged protein as detected with hexahistidine-binding antibody. The middle part demonstrates immunoreactivity of the same blot as the lower part upon stripping of hexahistidine-binding antibody and membrane reprobing with p25-1 antibody. This demonstrates that both the native and the acidic isoforms can bind AS aggregates. One of two similar experiments is shown. E, aggregates, assembled from full-length AS-(1–140) (A-140) and C terminally truncated AS-(1–95) (A-95), were incubated with rat brain cytosol (C), and their p25 binding was analyzed by sedimentation analysis. Input of cytosol and AS-(1–140) aggregates and pellet fractions of individual samples were analyzed for their p25 immunoreactivity (IR; upper panel) and silver-stainable proteins in 12–24-kDa range (lower panel). Molecular size markers in kDa are shown to the left in the lower panel. Aggregates assembled from AS lacking the C-terminal 45 residues cannot bind p25. One of three similar experiments is shown. F, rat brain p25 (p25) binds preferentially to aggregated AS. Negligible p25 amounts were present in the pellet upon sedimentation without aggregates (lane 1) and in the presence of monomeric AS (lane 9). In contrast, p25 with aggregates was present in lanes 2–8, and the amount of aggregate-associated p25 was not significantly inhibited upon co-incubation with increasing amounts of monomeric AS. Ratio of monomeric to aggregated AS (M/A) is indicated. One of three similar experiments is shown.

Characterization of Recombinant Human p25—The p25 protein was first identified as a bovine brain-specific phosphoprotein (25), and it has recently been attributed to tubulin-assembling properties (29). Sequence analysis demonstrates that p25 belongs to the highly conserved p25 gene family present in mammals, flies, nematodes, and even tetrahymenae (44). The human genome contains at least three p25-like genes, here designated as p25, p25, and p25, but previously named 25-kDa brain-specific protein (Swiss-Prot accession number O94811 [GenBank] ), brain-specific protein (Swiss-Prot accession number P59282 [GenBank] ), and protein CGI-38 (45, 46) (Swiss-Prot accession number Q9BW30), among which only the p25 protein has been detected. These three gene products display a high degree of sequence identity in their C termini, but their N termini differ, with the -form containing a unique 43-amino acid insertion (Fig. 2A).

View larger version (45K): [in this window] [in a new window]   FIG. 2. Cloning and expression of recombinant human p25. A, alignment of the amino acid sequence of the three human p25 gene products p25 (p25a), p25 (p25b), and p25 (p25g). -Specific N-terminal segment is marked by a line. This segment is deleted in the recombinant human p25-(3–43) protein used in this study. Amino acid sequence identity marked by gray boxes and insets are shown by vertical lines. Number of last amino acid in each line is shown to the right. B, left panel, purity of 5 µg of recombinant human p25 and deletion mutant p25-(3–43) assessed by Coomassie Blue staining after reducing SDS-PAGE. Right panel, p25 (2 µg) heated in SDS-loading buffer in the absence (Ox.) and presence of 20 mM dithioerythritol (Red.) and subjected to SDS-PAGE and silver staining. Molecular size markers in kDa for both panels indicated to the left. C, characterization of p25-1 antibody. Dilutions of recombinant p25 (lanes 1 and 2, 2 µg; lane 3, 0.4 µg; lane 4, 0.1 µg) and rat brain cytosol (lanes 5 and 6, 26 µg; lane 7, 13 µg; lane 8, 6 µg) resolved by reducing SDS-PAGE and by subjecting dilutions to immunoblotting using rabbit p25-1 IgG. Effect of pre-absorbing p25-1 IgG (9 µg/ml) with recombinant p25 (450 µg/ml) demonstrated in lanes 2 and 6. Molecular size markers in kDa indicated to the left. D, purified recombinant human p25 analyzed by far-UV CD spectroscopy under native (solid line) and denaturing conditions (dotted line, 5 M urea). Abscissa represents the wavelength and the ordinate the corresponding molar ellipticity. E, fluorescence emission spectra of p25 under native (solid line) and denaturing conditions in 5 M urea (dotted line). The abscissa shows wavelength and the ordinate the corresponding fluorescent emission. Experiments in D and E were performed on two preparations of p25.

p25 is a heat-stable protein, which would suggest that the protein is either (a) folded but very thermostable, or (b) natively unfolded. To gain structural insight into recombinant human p25, the purified protein was analyzed by far-UV CD spectroscopy (Fig. 2D). Deconvolution of the spectrum by the k2d program (kal-el.ugr.es/k2d/k2d.html) predicted 15% -helix, 30% -helix, and 55% random coil, but the fit was rather poor (data not shown), suggesting unusual features in its protein structure. However, p25 clearly has some degree of organized structure, because incubation of the protein in 5 M urea (well above the midpoint of denaturation, which is around 3.7 M urea, data not shown) caused a marked loss of spectral intensity (Fig. 2D). This was corroborated by the fluorescence spectrum, where the emission intensity peaks at 330 nm and undergoes a dramatic red shift to around 355 nm in the presence of 5 M urea that is typical of a buried Trp residue (Fig. 2E). The above data clearly indicate that human p25 possesses both a secondary and a tertiary structure, which are lost at high denaturant concentrations.

Characterization of the Interaction between -Synuclein and p25—The binding of brain p25 to AS aggregates (Fig. 1, A and B) could in principle be mediated via unidentified cytosolic linker proteins or require specific, yet concealed post-translational modifications of p25. Different experimental approaches were used to demonstrate a direct binding between purified recombinant human p25 and purified AS aggregates as follows: first, co-sedimentation analysis (Fig. 3A); second, immunoelectron microscopic demonstration of recombinant p25 decorating AS filaments (Fig. 3B); third, photoaffinity labeling of p25 by conjugates between AS aggregates and the ¹²⁵I-labeled cleavable cross-linker SASD (Fig. 3E); and fourth, competition analysis of the binding of ¹²⁵I-p25 to immobilized AS aggregates (Fig. 3F).

View larger version (37K): [in this window] [in a new window]   FIG. 3. Characterization of the direct binding of p25 to -synuclein. A, purified recombinant human p25 (p25) was incubated in the presence and absence of 10 µg of purified AS aggregates (A) and analyzed for co-sedimentation. p25 content in 10% of input and total pellet fractions was analyzed by immunoblotting. B, electron microscopic demonstration of p25 binding to AS filaments. AS filaments were incubated with recombinant p25, whereupon the p25-AS filament complexes were isolated by density gradient centrifugation. Purified filaments were incubated with p25-1 IgG (panel 1) and nonimmune IgG (panel 2), and subsequently anti-rabbit IgG was conjugated to 5-nm gold particles and analyzed by negative staining electron microscopy. A bar representing 100 nm is presented to the right. C, characterization of 125I-SASD/AS aggregate tracer by SDS-PAGE and autoradiography. 125I-SASD-AS aggregate (1,000,000 cpm) was either incubated in SDS-loading buffer without heating (lane 1); depolymerized by incubation in 4 M urea and 2% SDS overnight prior to addition of SDS-loading buffer and heating (lane 2); subjected to activation of photoreactive cross-linker prior to treatment as in lane 2 (lane 3); or subjected to activation of photoreactive cross-linker followed by reduction prior to treatment as in lane 2 (lane 4). UV designates photoactivation, red sample reduction. Molecular size markers in kDa presented to the left. D, specificity of photoaffinity labeling of ligands by 125I-SASD-AS aggregate tracer (1,000,000 cpm) demonstrated by incubating with FILA-1 IgG (6 µg), which binds AS aggregates, and nonimmune (N.I.) IgG (6 µg) as negative control. Samples were resolved by reducing SDS-PAGE. Left panel demonstrates protein stain of the gel by Coomassie Blue to ensure equal IgG. Right panel represents the 125I-labeling in the gel shown in the left panel as an autoradiograph. Cross-linker activation by UV light (UV) demonstrated below. Molecular size markers in kDa are presented to the left. E, photoaffinity labeling of wild type p25 (WT) and p25-(3–43) (3–43) by 125I-SASD/AS aggregates. 125I-SASD/AS aggregate tracer (1,000,000 cpm) was incubated with 3 µg of p25 (lanes 1, 2, 5, and 6) and p25-(3–43) (lanes 3, 4, 7, and 8) with and without activation of cross-linker by UV light. All samples were subjected to reducing SDS-PAGE, Coomassie Blue staining (left panel), and autoradiography (right panel). Activation of cross-linker by UV light (UV) demonstrated below. Molecular size markers in kDa presented to the left. F, quantitative analysis of 125I-p25 binding to immobilized AS-(1–140) aggregates. Purified AS aggregates were immobilized in microtiter plates and incubated with 50 pM 125I-p25 in the absence and presence of competitors; purified aggregates of AS-(1–140) (filled circle), AS-(1–125) (open circle), AS-(1–110) (filled triangle); monomeric AS-(1–140) (open triangle), -synuclein (filled diamond), a synthetic peptide corresponding to AS-(109–140) (open diamond), p25 (open square), and p25-(3–43) (closed square). Ordinate displays percentage of bound/free tracer and the abscissa the concentration of competitors. Points display mean ± 1 S.D. of triplicates from one of three representative experiments. Inset, p25 (3 µg) mixed with 10,000 cpm 125I-p25 and resolved by SDS-PAGE. Gel stained by Coomassie Blue (lane 1) and subjected to autoradiography (lane 2). Molecular size markers in kDa indicated to the left.

The 18.9-Å heterobifunctional cleavable cross-linker SASD was further employed to demonstrate the tight interaction between p25 and AS aggregates by photoaffinity labeling. The ¹²⁵I-labeled aggregates were quite resistant to depolymerization by short term incubation in SDS-PAGE loading buffer but could be depolymerized by prolonged incubation in 4 M urea, 2% SDS (Fig. 3C, lanes 1 versus 2) as demonstrated previously (34) for unlabeled AS aggregates. Activation of the photoreactive cross-linker by UV light made the AS aggregates insoluble in 4 M urea, 2% SDS (Fig. 3C, lane 3) because of the formation of intermolecular covalent bonds between different AS monomers in the aggregate. The SASD molecule contains a disulfide bridge, which, upon reduction, cleaves the cross-linker and leaves the ¹²⁵I label on the structure targeted by the photoreactive group. Hence, reduction of the labeled insoluble aggregates increased their solubility in urea/SDS and revealed the presence of ¹²⁵I-labeled AS monomers (Fig. 3C, lanes 3 versus 4). The labeled AS aggregates were used as a probe to label aggregate-binding ligands as demonstrated by the labeling of the AS aggregate-binding antibody FILA-1 but not the nonimmune IgG control (Fig. 3D, lanes 5 and 7). By this technique, both p25 and the deletion mutant p25-(3–43) were photoaffinity-labeled upon incubation with the ¹²⁵I-SASD conjugated AS aggregates (Fig. 3E, lanes 5 and 7).

A recently developed AS aggregate solid phase binding assay (34) allowed a detailed quantitative analysis of the binding of p25 to AS aggregates (Fig. 3F). The ¹²⁵I-labeled p25 tracer migrated predominantly as a single 25-kDa band, which co-migrated with Coomassie Blue-stained recombinant p25 (Fig. 3F, inset, lane 2 versus 1). The binding of 50 pM ¹²⁵I-p25 to immobilized aggregated AS was inhibited by fluid phase AS aggregates with an IC₅₀ of about 10 nM and to the same extent as when using unlabeled p25 and p25-(3–43) (Fig. 3D). Monomeric AS displayed an 10-fold higher IC₅₀ than aggregated AS (Fig. 3F). However, the molar concentration of the aggregates could not be expressed precisely due to their heterogeneous homopolymeric nature, and they are accordingly expressed by the concentration of their monomeric content. The selectivity of p25 for aggregated AS on a molar basis is hence even larger, and it is likely to lie in the range of 10²–10³, given that the aggregates contain 20–100 monomeric subunits. Accordingly, p25 binds to monomeric AS but exhibits a higher affinity for aggregated AS.

The basis for the aggregate selectivity within the AS primary structure was investigated using aggregates prepared from AS molecules truncated for the last 30 residues AS-(1–110) and 15 residues AS-(1–125). To ensure equal concentrations of the aggregates prior to analysis, samples from the purified aggregate stock solutions were depolymerized and subsequently compared by Coomassie Blue staining of SDS-PAGE (data not shown). AS-(1–110) aggregates displayed a 100-fold higher IC₅₀ than the wild type peptide (Fig. 3F), whereas the IC₅₀ for binding to AS-(1–125) was indistinguishable from the wild type AS. Moreover, -synuclein, whose C terminus resembles AS in terms of content and spacing of their negatively charged residues, and a synthetic peptide AS-(109–140), corresponding to AS C terminus, were unable to inhibit the tracer binding (Fig. 3F). The high affinity binding of p25 to AS aggregates accordingly relies on the presentation of a segment within the AS C terminus close to residues 110–125 in the context of an AS aggregate.

p25 Stimulates -Synuclein Aggregation—The binding of p25 to monomeric AS raised the possibility that p25 may affect the process of AS aggregation. This question was first investigated by an aggregate-sedimentation assay where AS was incubated in the absence and presence of substoichiometric concentrations of p25 followed by isolation of the AS aggregates by centrifugation (Fig. 4A). At time 0, a small amount of AS was detected in the pellet, which represents the background level of the assay when using 340 µM AS. A slight, spontaneous aggregation of AS was detected after 72 h of incubation. However, co-incubation of the AS with 3 and 10 µM recombinant human p25 produced a dose-dependent increase in the amount of aggregated AS. p25 was recovered in the pellet with the AS aggregates (Fig. 4A) but remained in the supernatant in the absence of AS (data not shown). p25 also stimulated the sedimentation of AS peptides carrying either of the two PD-causing mutations A30P and A53T (data not shown).

View larger version (44K): [in this window] [in a new window]   FIG. 4. p25 stimulates -synuclein aggregation. A, AS (340 µM) was incubated in the absence and presence of p25 (3.4 µM (1%) and 10 µM (3%)) at 37 °C for 3 days. The aggregational state of AS was determined by density gradient centrifugation. Input and insoluble pellets at days 0 and 3 were analyzed by reducing SDS-PAGE. Positions of AS and p25 are indicated to the left. B, AS was incubated in the absence (filled circles) and presence of 1% (open circles) and 3% (filled triangles) p25 at 37 °C, and samples were recovered for analysis of thioflavin-T (TT) fluorescence at days 0, 1, and 3. Left panel, 100 µM AS; middle panel, 200 µM AS; and right panel, 340 µM AS. Ordinate represents TT fluorescence in arbitrary units. Note the different scale of the three panels. Points represent mean of triplicates. One of five representative experiments is presented. C, AS (340 µM) aggregation performed for 3 days in the absence and presence of 1% p25. Filaments from five individual samples were purified by density gradient centrifugation, resuspended in PBS, applied onto grids for quantitative negative stain electron microscopy, and analyzed in systematic random measuring mode. Upper and lower left panels demonstrate representative electron micrographs obtained in the absence and presence of p25.A1-µm bar in the lower left panel applies to the high magnification images in the upper and lower left panels, and 5-µm bars are present in the low magnification images in the middle upper and lower images. Right panel shows grid percentage covered with filaments in the absence (AS) and presence of 1% p25 (AS + p25). There is a significant difference (p = 1.2 x 10–4) in area fraction of filaments between negatively stained samples of AS and AS incubated with 1% recombinant p25 as visualized by electron microscopy.

p25 Accumulates in Pathological -Synuclein Inclusions—In the rat brain, p25 is distributed to cells with a morphology corresponding to that of oligodendrocytes and choroid plexus epithelial cells (data not shown). This is consistent with early observations in rat (26). We had the impression that virtually all oligodendrocytes present in both gray and white matter regions expressed p25. Similarly, in human brains the vast majority of oligodendrocytes was also immunopositive for p25. The immunoreaction product within oligodendrocytes of rat brain and human control cases distributed predominantly to the perinuclear cytoplasm leaving their nuclei unlabeled, whereas peripheral processes displayed a more punctate pattern evident both by histochemical staining and immunofluorescence microscopy (Fig. 5, A and B). Cellular labeling of oligodendrocytes was never observed when the primary antibody was omitted from the immunoreaction (Fig. 5C). MSA cases, however, showed a population of oligodendrocytes with a p25 distribution in the perinuclear cytoplasm (Fig. 5E, arrowhead) that was more robust than the slight labeling of the perinuclear cytoplasm in the oligodendrocytes (Fig. 5E, arrows) that resembled the controls. The p25 distribution appeared to be expanded in the group of oligodendrocytes with the most intense p25 immunoreactivity. A parallel to the cellular distribution of AS in oligodendrocytes during pathological conditions was made in the brains of MSA cases in which oligodendrocytes are known to express AS in glial cytoplasmic inclusions (3, 47). AS indeed labeled several oligodendrocytes of MSA (Fig. 5D), leaving oligodendrocytes of PD, LBD, and normal cases unstained (data not shown). The use of double-labeling fluorescence and confocal microscopy revealed a clear co-distribution of p25 and AS in many oligodendrocytes (Fig. 5, G–I). Quantification of images like Fig. 5, G and H, revealed that about 60% of the AS-positive inclusions were p25-positive. The number of AS-positive oligodendrocytes was, however, significantly lower compared with the number of oligodendrocytes labeled with p25.

View larger version (126K): [in this window] [in a new window]   FIG. 5. p25 and -synuclein are co-expressed in glial cytoplasmic inclusions in multiple system atrophy. A, p25 in human oligodendrocytes of the normal brain white matter. Note the labeling of the thin perinuclear cytoplasm (arrow). B, p25 in human oligodendrocytes of the normal brain white matter shown at high power magnification. C, omitting the primary anti-p25 antibody of the immunoreaction abolishes labeling of oligodendrocytes. D, AS in human oligodendrocytes of multiple system atrophy case. Section shows labeled oligodendrocytes (arrowhead) of white matter underlying cerebral cortex. Subcellular AS distribution within individual oligodendrocytes is heterogeneous but leaves the nucleus unlabeled. E, p25 in human oligodendrocytes of same multiple system atrophy case. Numerous labeled oligodendrocytes are seen. Subcellular p25 distribution within individual oligodendrocytes is heterogeneous but leaves the nucleus unlabeled. It varied from labeling of thin perinuclear cytoplasm in normal and apparently normal oligodendrocytes (arrow) to a robust labeling of expanded cytoplasm in degenerating cells (arrowhead). F, p25 in human oligodendrocytes of multiple system atrophy case shown at high power magnification highlighting principal difference in subcellular p25 distribution within apparently normal (arrow) and pathological (arrowhead) oligodendrocytes. G–I, double-labeling fluorescence revealing co-distribution of AS (G) and p25 (H) in glial cytoplasmic inclusions (arrowhead in D and E) of multiple system atrophy case. I, superimposing the images reveals co-localization of p25 and AS in oligodendrocytes. H and I, presumably normal oligodendrocytes without glial cytoplasmic inclusions identified as p25-labeling of thin perinuclear cytoplasmic region (arrows in E) and devoid of AS immunoreactivity (arrows in I). Scale bars = 10 µm (A, C–E, and G–I), 2.5 µm (B and F).

View larger version (91K): [in this window] [in a new window]   FIG. 6. p25 in neuronal Lewy bodies. A, p25 in neurons of supraoptic neurons of rat brain. Inset shows labeled nucleus at low power magnification. At larger magnification, the evident labeling of both supraoptic neurons (arrowheads) and oligodendrocytes of optic tract (arrows) is shown. B, p25 labeling of pigmented neuron of human substantia nigra pars compacta is shown at high power magnification. p25 distributes to the periphery of the Lewy body. C–E, double-labeling fluorescence revealing co-distribution of AS (C) and p25 (D) in the Lewy body of human substantia nigra pars compacta. Superimposing the images reveals co-localization of p25 and AS in Lewy body (E). F–H, control of specificity of p25 immunostaining. Lewy body containing neurons was labeled as in C–E, but the primary antibody was omitted in G. Scale bars = 40 µm(A), 20 µm(B), 20 µm(C–E), and 10 µm(F–H).

The emergence of procedures to isolate AS-containing inclusions from human PD, MSA, and LBD tissue have allowed detailed structural and biochemical analyses of these structures. Fig. 7, A and B, demonstrates that p25 is present in both Lewy bodies and Lewy neurites isolated from LBD brain tissue, and the p25 in the inclusions co-localizes to a large extent with the AS as demonstrated by double immunofluorescence confocal laser-scanning microscopy. The negative controls without primary antibodies showed no staining (data not shown) as demonstrated for the tissue staining with the same antibodies in Fig. 6G. 100% of the AS-positive isolated glial cytoplasmic inclusions and 80% of isolated AS-positive Lewy bodies and Lewy neurites were p25-positive. Biochemical analysis revealed that p25 in both isolated Lewy bodies and glial cytoplasmic inclusions appeared as a single 27-kDa band with no signs of aggregation and degradation when analyzed by reducing SDS-PAGE and immunoblotting (Fig. 7C).

View larger version (20K): [in this window] [in a new window]   FIG. 7. -Synuclein and p25 co-localize in Lewy bodies and Lewy neurites isolated from Lewy body dementia brain tissue. Lewy bodies (A) and Lewy neurites (B) isolated from LBD brain tissue were analyzed for localization of -synuclein (a-syn) and p25 (P25) by confocal laser-scanning microscopy. AS immunoreactivity, detected with sheep anti--synuclein antibody, is presented in the left columns, and p25, detected by the rabbit p25-1 IgG, is presented in the middle columns. Merged images are presented in the right columns. 10-µm scale bars are presented. C, immunoblot analysis of p25 in isolated brain inclusions. Extracts of human brain tissue homogenate (BH) (20 µg; lane 1), purified Lewy bodies (LB) (5 µg) from brain tissue affected by LBD (lane 2), and isolate