Detecting Morphologically Distinct Oligomeric Forms of α-Synuclein
2009; Elsevier BV; Volume: 284; Issue: 17 Linguagem: Inglês
10.1074/jbc.m806559200
ISSN1083-351X
AutoresSharareh Emadi, Srinath Kasturirangan, Min S. Wang, Philip Schulz, Michael R. Sierks,
Tópico(s)Nerve injury and regeneration
ResumoNeuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of α-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of α-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of α-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of α-synuclein in vitro, blocks extracellular α-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue. Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of α-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of α-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of α-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of α-synuclein in vitro, blocks extracellular α-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue. Parkinson disease (PD) 2The abbreviations used are: PD, Parkinson disease; scFv, single chain antibody fragment; AFM, atomic force microscope; ThT, Thioflavin T; AD, Alzheimer disease; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; LDH, lactic acid dehydrogenase; BSA, bovine serum albumin; ND, no disease; MFI, mean fluorescence intensity. 2The abbreviations used are: PD, Parkinson disease; scFv, single chain antibody fragment; AFM, atomic force microscope; ThT, Thioflavin T; AD, Alzheimer disease; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; LDH, lactic acid dehydrogenase; BSA, bovine serum albumin; ND, no disease; MFI, mean fluorescence intensity. is the second most common neurodegenerative disorder of the elderly, affecting more than 500,000 people in the United States (1.Price D.L. Nature. 1999; 399: A3-A5Crossref PubMed Scopus (67) Google Scholar), with 50,000 new cases reported each year at an annual cost estimated at 10 billion dollars per year. Pathologically, PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and formation of fibrillar cytoplasmic inclusions known as Lewy bodies and Lewy neurites (2.Schapira A.H. Baillieres Clin. Neurol. 1997; 6: 15-36PubMed Google Scholar, 3.Gomez-Tortosa E. Newell K. Irizarry M.C. Albert M. Growdon J.H. Hyman B.T. Neurology. 1999; 53: 1284-1291Crossref PubMed Google Scholar). The protein α-synuclein has been strongly linked to PD (4.Spillantini M.G. Crowther R.A. Jakes R. Cairns N.J. Lantos P.L. Goedert M. Neurosci. Lett. 1998; 251: 205-208Crossref PubMed Scopus (795) Google Scholar, 5.Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Am. J. 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Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1233) Google Scholar). The central region, non-amyloid component, is extremely hydrophobic and includes a 12-residue stretch (VTGVTAVAQKTV) that is essential for aggregation (15.Serpell L.C. Berriman J. Jakes R. Goedert M. Crowther R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4897-4902Crossref PubMed Scopus (636) Google Scholar). The C-terminal region is enriched with acidic glutamate and aspartate residues and is responsible for the chaperone function of α-synuclein (16.Volles M.J. Lee S.J. Rochet J.C. Shtilerman M.D. Ding T.T. Kessler J.C. Lansbury Jr., P.T. Biochemistry. 2001; 40: 7812-7819Crossref PubMed Scopus (604) Google Scholar). α-Synuclein normally exists as an unfolded protein, but it can adopt several different folded conformations depending on the environment, including small aggregates or oligomers, spherical and linear protofibrils, as well as the fibrillar structure found in Lewy bodies (14.Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1233) Google Scholar, 15.Serpell L.C. Berriman J. Jakes R. Goedert M. Crowther R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4897-4902Crossref PubMed Scopus (636) Google Scholar). A growing body of evidence implicates the oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death (16.Volles M.J. Lee S.J. Rochet J.C. Shtilerman M.D. Ding T.T. Kessler J.C. Lansbury Jr., P.T. Biochemistry. 2001; 40: 7812-7819Crossref PubMed Scopus (604) Google Scholar, 17.Volles M.J. Lansbury Jr., P.T. 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However, recent studies have suggested that α-synuclein also has extracellular pathogenic effects (22.Borghi R. Marchese R. Negro A. Marinelli L. Forloni G. Zaccheo D. Abbruzzese G. Tabaton M. Neurosci. Lett. 2000; 287: 65-67Crossref PubMed Scopus (299) Google Scholar, 23.El-Agnaf O.M. Salem S.A. Paleologou K.E. Curran M.D. Gibson M.J. Court J.A. Schlossmacher M.G. Allsop D. FASEB J. 2006; 20: 419-425Crossref PubMed Scopus (553) Google Scholar, 24.Lee P.H. Lee G. Park H.J. Bang O.Y. Joo I.S. Huh K. J. Neural Transm. 2006; 113: 1435-1439Crossref PubMed Scopus (174) Google Scholar, 25.Tokuda T. Salem S.A. Allsop D. Mizuno T. Nakagawa M. Qureshi M.M. Locascio J.J. Schlossmacher M.G. El-Agnaf O.M. Biochem. Biophys. Res. Commun. 2006; 349: 162-166Crossref PubMed Scopus (355) Google Scholar). α-Synuclein was detected in blood plasma and cerebrospinal fluid in both monomeric and oligomeric forms (22.Borghi R. Marchese R. Negro A. Marinelli L. Forloni G. Zaccheo D. Abbruzzese G. Tabaton M. 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Chem. 2001; 276: 27441-27448Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 32.Emadi S. Barkhordarian H. Wang M.S. Schulz P. Sierks M.R. J. Mol. Biol. 2007; 368: 1132-1144Crossref PubMed Scopus (135) Google Scholar). Despite all these studies, it is still not clear how the various aggregate forms of α-synuclein are involved in the progression of PD. Therefore, reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only for fundamental studies of how α-synuclein aggregates affect cell function but also as potential diagnostic and therapeutic agents for PD. Recently, we reported inhibition of both aggregation and extracellular toxicity of α-synuclein in vitro by a single chain variable domain antibody fragment (scFv) that specifically recognized an oligomeric form of α-synuclein (32.Emadi S. Barkhordarian H. Wang M.S. Schulz P. Sierks M.R. J. Mol. Biol. 2007; 368: 1132-1144Crossref PubMed Scopus (135) Google Scholar). In this study, we describe a second scFv (syn-10H) that binds a larger later stage oligomeric form of α-synuclein than the previously reported scFv. The syn-10H scFv neutralizes α-synuclein-induced toxicity in both undifferentiated and differentiated SH-SY5Y human neuroblastoma cell line and inhibits α-synuclein aggregation in vitro. The syn-10H scFv reacts specifically with homogenized PD brain tissue but does not cross-react with similarly treated samples taken from Alzheimer disease (AD) or healthy brain samples. Such scFvs therefore have potential value as diagnostic reagents to identify the presence of specific oligomeric species in PD tissue and fluid samples. The scFvs also have value as therapeutic agents as they can be used either extracellularly or expressed intracellularly (intrabodies) to prevent formation of toxic aggregates in vivo whether inside or outside of cells. Intrabodies have been used efficiently to neutralize toxic effects of different pathogenic agents, including α-synuclein (33.Wheeler Y.Y. Chen S.Y. Sane D.C. Mol. Ther. 2003; 8: 355-366Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 34.Murphy R.C. Messer A. Brain Res. Mol. Brain Res. 2004; 121: 141-145Crossref PubMed Scopus (54) Google Scholar, 35.Lecerf J.M. Shirley T.L. Zhu Q. Kazantsev A. Amersdorfer P. Housman D.E. Messer A. Huston J.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4764-4769Crossref PubMed Scopus (182) Google Scholar, 36.Zhou C. Emadi S. Sierks M.R. Messer A. Mol. Ther. 2004; 10: 1023-1031Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Moreover, immunization studies in mouse models of PD have shown that extracellular antibodies can reduce accumulation of intracellular aggregates of α-synuclein (37.Masliah E. Rockenstein E. Adame A. Alford M. Crews L. Hashimoto M. Seubert P. Lee M. Goldstein J. Chilcote T. Games D. Schenk D. Neuron. 2005; 46: 857-868Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar), thereby providing precedent for the use of scFvs in potential passive vaccination strategies for treating PD. The Tomlinson I and J phage libraries, helper phage KM13, Escherichia coli TG1, and HB2151 were obtained from Medical Research Council (Cambridge, UK). Each phage library has a diversity of greater than 108 and are comprised of a single polypeptide with the VH and VL domains connected by a flexible (Gly4Ser)3 linker. The phage library was propagated essentially as described (32.Emadi S. Barkhordarian H. Wang M.S. Schulz P. Sierks M.R. J. Mol. Biol. 2007; 368: 1132-1144Crossref PubMed Scopus (135) Google Scholar, 38.Barkhordarian H. Emadi S. Schulz P. Sierks M.R. Protein Eng. Des. Sel. 2006; 19: 497-502Crossref PubMed Scopus (45) Google Scholar). Libraries were grown separately and mixed in equal titers for the panning experiments. α-Synuclein plasmid provided by Dr. Michael J. Volles (Brigham and Women's Hospital, Harvard Medical School) was transformed into BL-21 competent cells, plated onto LB-agar plates (supplemented with 100 μg/ml ampicillin), and grown overnight at 37 °C. Single colonies of BL21 (DE3) were grown and purified essentially as described (39.Volles M.J. Lansbury Jr., P.T. J. Mol. Biol. 2007; 366: 1510-1522Crossref PubMed Scopus (164) Google Scholar). α-Synuclein was lyophilized and stored at −80 °C until further use. The lyophilized α-synuclein stock was dissolved in Buffer A (25 mm Tris-HCl and 150 mm NaCl, pH 7.4) to a concentration of 70 μm. Oligomeric aggregates of α-synuclein were obtained by incubating at 37 °C for 7–10 days without shaking. Fibrils were obtained upon longer incubation up to 35 days. Aβ40 oligomers (BIOSOURCE) were dissolved in 100% 1,1,1,3,3,3-hexafluoro-2-propanol at a concentration of 1 mg/ml in 250-μl aliquots, air-dried, and stored at −20 °C until further use. Aliquots were resuspended in 100% dimethyl sulfoxide (DMSO) and further diluted in Buffer A to a 20 μm concentration and incubated at 37 °C without shaking for 5 days. The oligomeric morphologies of Aβ40 were verified by atomic force microscopy (AFM) prior to study (40.Zameer A. Kasturirangan S. Emadi S. Nimmagadda S.V. Sierks M.R. J. Mol. Biol. 2008; 384: 917-928Crossref PubMed Scopus (66) Google Scholar). A diluted sample of α-synuclein (20 μm) containing predominantly larger aggregate forms was used for bio-panning studies. Three rounds of bio-panning against the α-synuclein mixture were performed essentially as described (41.Marks J.D. Hoogenboom H.R. Bonnert T.P. McCafferty J. Griffiths A.D. Winter G. J. Mol. Biol. 1991; 222: 581-597Crossref PubMed Scopus (1420) Google Scholar). Briefly, immunotubes (Maxisorb, Nunc) were coated overnight at 4 °C with 20 μm sample of α-synuclein mixture in 50 mm carbonate/bicarbonate, pH 9.6. The tube was then blocked with 3% BSA in phosphate-buffered saline (PBS: 10 mm phosphate and 150 mm NaCl, pH 7.4) for 2 h at 37 °C. An aliquot of phage library (1012 plaque-forming unit/ml) in 4 ml of 1% BSA/PBS was added to the immunotube and incubated for 30 min with continuous rocking, followed by 90 min without rocking at 22 °C. The sample was subsequently washed 10 times with 1 ml of PBS/Tween 0.1% and 15 times with PBS to remove the nonspecifically bound phage. The bound phage were eluted from the immunotube by incubation with triethylamine (100 mm, pH 11) for 10 min, followed by neutralization with Tris-HCl (1 m, pH 7.4) and hydrolysis with trypsin (1 mg/ml) and calcium (1 mm) for 30 min with continuous rocking at room temperature. A 1-ml aliquot of eluted phage was added to 10 ml of E. coli TG1 (A600 of 0.4) and incubated for 30 min at 37 °C. Eluted phage were amplified by infecting fresh TG1 cells in the presence of helper phage KM13 (5 × 1010) and purified according to standard protocols (Cambridge, UK). Phage titers were determined by serial dilutions on agar plates containing ampicillin (100 μg/ml). Polyclonal ELISA—The polyclonal phage ELISA was performed as described previously (38.Barkhordarian H. Emadi S. Schulz P. Sierks M.R. Protein Eng. Des. Sel. 2006; 19: 497-502Crossref PubMed Scopus (45) Google Scholar). High binding polystyrene microtiter plates were coated with 5 μm of the α-synuclein mixture in carbonate/bicarbonate, pH 9.6, and 1010 plaque-forming unit aliquots of PEG-precipitated phage was added. Bound phage was detected after 1 h of incubation with a 1:5000 dilution of anti-M13 antibody horseradish peroxidase (HRP) conjugate and detected with HRP substrate 3,3′,5,5′-tetramethyl-benzidine (Sigma) after 20 min. The activity was determined by subtracting A650 from A450 using a Wallac 1420 plate reader (PerkinElmer Life Sciences). Monoclonal ELISA—Individual clones obtained from panning against α-synuclein mixture were grown as described (32.Emadi S. Barkhordarian H. Wang M.S. Schulz P. Sierks M.R. J. Mol. Biol. 2007; 368: 1132-1144Crossref PubMed Scopus (135) Google Scholar). The high binding microtiter plates (Corning Glass) were coated with 5 μm α-synuclein mixture and blocked as described above. Bacterial supernatant containing antibody fragments was added to each well (100 μl/well). Bound phage were detected as described above. Soluble scFv was produced by expressing recovered phagemid samples in the nonsuppressor E. coli strain HB2151 (42.Carter P. Bedouelle H. Winter G. Nucleic Acids Res. 1985; 13: 4431-4443Crossref PubMed Scopus (410) Google Scholar). Individually selected clones were grown as described previously (32.Emadi S. Barkhordarian H. Wang M.S. Schulz P. Sierks M.R. J. Mol. Biol. 2007; 368: 1132-1144Crossref PubMed Scopus (135) Google Scholar), and scFv production was induced by the addition of 1 mm isopropyl β-d-thiogalactopyranoside. Supernatant and periplasmic fractions were assayed for antigen binding by ELISA as described (32.Emadi S. Barkhordarian H. Wang M.S. Schulz P. Sierks M.R. J. Mol. Biol. 2007; 368: 1132-1144Crossref PubMed Scopus (135) Google Scholar). High affinity microtiter plates were coated with 5 μm α-synuclein mixture. After blocking and washing, an aliquot (100 μl) of supernatant and periplasmic fractions containing antibody fragments was added to each well, and the plate was incubated for 2 h at room temperature. Bound antibodies were detected after a 1-h incubation using 1:500 dilution of anti-c-Myc tag (9E10) HRP conjugate (Santa Cruz Biotechnology). Plasmid was isolated from E. coli TG1 using a plasmid mini-prep kit (Qiagen, Valencia, CA) according to the manufacturer's protocols. The presence of a full-length 935-bp scFv insert was confirmed by PCR as described (32.Emadi S. Barkhordarian H. Wang M.S. Schulz P. Sierks M.R. J. Mol. Biol. 2007; 368: 1132-1144Crossref PubMed Scopus (135) Google Scholar, 43.Hoogenboom H.R. Winter G. J. Mol. Biol. 1992; 227: 381-388Crossref PubMed Scopus (388) Google Scholar). The false stop codon (TGA) in the constant region of scFv was corrected and replaced with a tryptophan (TGG) using site-directed mutagenesis as described (44.Marcus W.D. Wang H. Lohr D. Sierks M.R. Lindsay S.M. Biochem. Biophys. Res. Commun. 2006; 342: 1123-1129Crossref PubMed Scopus (18) Google Scholar). Selected individual clones were purified using a protein A-Sepharose column (GE Healthcare) as described previously (32.Emadi S. Barkhordarian H. Wang M.S. Schulz P. Sierks M.R. J. Mol. Biol. 2007; 368: 1132-1144Crossref PubMed Scopus (135) Google Scholar). Lyophilized α-synuclein was dissolved to a final concentration of 70 μm in Buffer A, filtered through a 0.2-μm filter, and incubated at 37 °C for 35 days. Aliquots (3 μl) of incubated solution taken at different time points were applied to nitrocellulose membranes (Bio-Rad) and air-dried. The membrane was blocked with phosphate-buffered saline containing 2% milk (PMSM) and probed with 0.3 mg/ml syn-10H for 2 h followed by overnight incubation with 1:500 dilution of 9E10. The immunoreactivity was detected after a 1-h incubation using a 1:1000 dilution of anti-mouse HRP. Lyophilized α-synuclein was dissolved to a final concentration of 100 μm in Buffer A and incubated at 37 °C for 7 days without shaking. An aliquot (20 μl) of incubated solution was analyzed by SDS-PAGE using a Tris/Tricine buffer system (45.Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10410) Google Scholar) and developed using silver staining according to the manufacturer's protocols. The sample was then transferred to a nitrocellulose membrane as described (32.Emadi S. Barkhordarian H. Wang M.S. Schulz P. Sierks M.R. J. Mol. Biol. 2007; 368: 1132-1144Crossref PubMed Scopus (135) Google Scholar) and probed with 0.3 mg/ml syn-10H as described above. α-Synuclein was dissolved to a final concentration of 70 μm in Buffer A, filtered as described above, and incubated at 37 °C either with or without the addition of 15 μm scFv. Aggregation of α-synuclein was measured in triplicate at various time points as described previously (32.Emadi S. Barkhordarian H. Wang M.S. Schulz P. Sierks M.R. J. Mol. Biol. 2007; 368: 1132-1144Crossref PubMed Scopus (135) Google Scholar, 46.Liu R. Yuan B. Emadi S. Zameer A. Schulz P. McAllister C. Lyubchenko Y. Goud G. Sierks M.R. Biochemistry. 2004; 43: 6959-6967Crossref PubMed Scopus (114) Google Scholar). Human neuroblastoma cells (SH-SY5Y) were maintained in culture flasks in medium containing 50% (v/v) minimal essential medium, 50% (v/v) Ham's modification of F-12, 10% (v/v) fetal bovine serum, 1% (w/v) l-glutamine (3.6 mm), and 1% penicillin/streptomycin antibiotic and grown in a 5% CO2 atmosphere at 37 °C. Cells were harvested from flasks and plated in 48-well polystyrene plates (Corning Glass) with 3 × 105 cells per 300 μl of medium per well. The cells were differentiated by adding 10 μm retinoic acid to each well and incubating at 37 °C for 4 days. As reported previously, SH-SY5Y cells treated with such a protocol exhibit several characteristics of cholinergic neurons, including expression of ChAT and VMAT (47.Pahlman S. Hoehner J.C. Nanberg E. Hedborg F. Fagerstrom S. Gestblom C. Johansson I. Larsson U. Lavenius E. Ortoft E. Söderholm H. Eur. J. Cancer. 1995; 31A: 453-458Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 48.Presgraves S.P. Ahmed T. Borwege S. Joyce J.N. Neurotox. Res. 2004; 5: 579-598Crossref PubMed Scopus (209) Google Scholar). After 4 days, the medium was exchanged with 300 μl of serum-free media, followed by the addition of the preincubated mixtures of α-synuclein with or without scFv to individual wells. The same volume of medium was added to the control cultures. Plates were then incubated at 37 °C for 48 h. Cell viability was measured by lactic acid dehydrogenase (LDH) assay. Briefly, cells were centrifuged, and aliquots (50 μl) of the media from each well were transferred to a 96-well plate. The supplied buffer and substrate were then added to the supernatant as described by the manufacturer. Absorbance was measured by subtracting A650 from A480 using a Wallac 1420 plate reader. LDH release was determined by dividing the absorbance of treated wells by the absorbance of untreated wells. The data are reported as percentage of control value obtained from three independent experiments. The cell viability was also measured using trypan blue assay as described previously (32.Emadi S. Barkhordarian H. Wang M.S. Schulz P. Sierks M.R. J. Mol. Biol. 2007; 368: 1132-1144Crossref PubMed Scopus (135) Google Scholar). SH-SY5Y cells were detached from flasks using trypsin, washed with PBS, and plated in a 96-well plate with the density of 2 × 104 cells per well per 100 μl of PBS. The cell was centrifuged, and the pellet was washed twice with buffer containing PBS and BSA (0.5%). The cells were then labeled with or without syn-10H scFv (0.3 mg/ml) for 1 h at 4 °C. After washing, the pellet was incubated with 9E10 antibody (Roche Applied Science) for 1 h at 4 °C followed by the addition of fluorescein conjugated goat anti-mouse antibody (Invitrogen). The percentage and mean fluorescence intensity (MFI) of positive cells were analyzed with a flow cytometer (FACSCalibur system). Control samples stained with secondary antibody alone were included in each experiment. Topographic AFM images were obtained in air at room temperature using a Tapping Mode AFM with a Nanoscope IIIa controller (Veeco, Santa Barbara, CA). Images were acquired using oxide sharpened Si3N4 AFM tips (k = 40 n/m, ∼300-kHz) (model OTESPA, Veeco, Santa Barbara, CA) at scan rates of 2–3 Hz and at scan resolution of 512 samples per line. AFM images were analyzed with the scanning probe imaging processor software (Image Metrology) to generate height distribution histograms for each sample. A 600 × 7.8-mm BioSep-SEC S-2000 column (Phenomenex) on a System Gold high pressure liquid chromatograph (Beckman Coulter) was washed and equilibrated with PBS. An aliquot of purified α-synuclein (100 μm) was incubated in Buffer A at 37 °C for 7 days and was run on the size exclusion column at a 0.8 ml/min flow rate. After washing the column, 0.5-ml fractions were collected and analyzed by electrophoresis on a 10% Tris/Tricine SDS-PAGE (45.Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10410) Google Scholar). Human brain sections were a generous donation from Dr. Thomas Beach (Sun Health Research Institute, Sun City, AZ). The human brain tissue samples were homogenized in a buffer containing 50 mm Tris-HCl, pH 7.5, 5 mm EDTA, and 1% SDS supplemented with protease inhibitor mixture (Sigma). The samples were sonicated on ice using 10-s bursts followed by 15-s pauses for a total of 5 min using a Fisher sonic dismembrator. Homogenates were centrifuged for 10 min at 13,000 rpm at 4 °C. Supernatants of brain homogenates were aliquoted and stored at −80 °C until further use. A3-μl aliquot of brain sample was applied to a nitrocellulose membrane and air-dried. The me
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