Quantitative Proteomics Identifies Surfactant-Resistant α-Synuclein in Cerebral Cortex of Parkinsonism-Dementia Complex of Guam but Not Alzheimer's Disease or Progressive Supranuclear Palsy
2007; Elsevier BV; Volume: 171; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.070015
ISSN1525-2191
AutoresWan Jou Yang, Randall L. Woltjer, Izabela Sokal, Catherine Pan, Yan Wang, Mary Brodey, Elaine R. Peskind, James B. Leverenz, Jing Zhang, Daniel P. Perl, Douglas Galasko, Thomas J. Montine,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoParkinsonism-dementia complex (PDC) remains a significant health burden to the Chamorro population. We tested the hypothesis that quantitative proteomics might provide fresh insight into this enigmatic illness by analyzing proteins resistant to surfactant extraction from patients with Alzheimer's disease (AD) or PDC and their matched controls using isobaric tags for relative and absolute quantification. In addition to the expected increase in abnormal frontal cortical Aβ peptides, tau, ubiquitin, and apolipoprotein E in AD, and tau in PDC, we identified α-synuclein (SNCA) as a major abnormal protein in PDC but not AD. We confirmed our isobaric tags for relative and absolute quantification findings by enzyme-linked immunosorbent assay in frontal and temporal cortices. We extended our assays to include a limited number of cases of progressive supranuclear palsy (PSP) and dementia with Lewy bodies; we observed increased abnormal tau but not SNCA in PSP, and abnormal SNCA in dementia with Lewy bodies that was quantitatively similar to PDC. Finally, soluble Aβ oligomers were selectively increased in AD but not PDC or PSP. These results show that frontal and temporal cortex in PDC is distinguished from AD and PSP by its accumulation of abnormal SNCA and suggest that PDC be considered a synucleinopathy as well as a tauopathy. Parkinsonism-dementia complex (PDC) remains a significant health burden to the Chamorro population. We tested the hypothesis that quantitative proteomics might provide fresh insight into this enigmatic illness by analyzing proteins resistant to surfactant extraction from patients with Alzheimer's disease (AD) or PDC and their matched controls using isobaric tags for relative and absolute quantification. In addition to the expected increase in abnormal frontal cortical Aβ peptides, tau, ubiquitin, and apolipoprotein E in AD, and tau in PDC, we identified α-synuclein (SNCA) as a major abnormal protein in PDC but not AD. We confirmed our isobaric tags for relative and absolute quantification findings by enzyme-linked immunosorbent assay in frontal and temporal cortices. We extended our assays to include a limited number of cases of progressive supranuclear palsy (PSP) and dementia with Lewy bodies; we observed increased abnormal tau but not SNCA in PSP, and abnormal SNCA in dementia with Lewy bodies that was quantitatively similar to PDC. Finally, soluble Aβ oligomers were selectively increased in AD but not PDC or PSP. These results show that frontal and temporal cortex in PDC is distinguished from AD and PSP by its accumulation of abnormal SNCA and suggest that PDC be considered a synucleinopathy as well as a tauopathy. 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Classic biochemical approaches in the 1980s and early 1990s established that increased accumulation of abnormally hydrophobic protein, defined by their insolubility when extracted with surfactants (often referred to as detergents) such as Triton X-100 (Triton-insoluble or TI) or N-lauroylsarcosine (sarkosyl-insoluble or SI), as a characteristic feature of several neurodegenerative diseases. Indeed, the proteins resistant to surfactant extraction were identified as the major components of hallmark lesions in AD: amyloid β (Aβ) peptides in senile plaques and abnormally phosphorylated tau in neurofibrillary tangles (NFTs).28Wong CW Quaranta V Glenner GG Neuritic plaques and cerebrovascular amyloid in Alzheimer disease are antigenically related.Proc Natl Acad Sci USA. 1985; 82: 8729-8732Crossref PubMed Scopus (339) Google Scholar, 29Lee VM Balin BJ Otvos Jr, L Trojanowski JQ A68: a major subunit of paired helical filaments and derivatized forms of normal tau.Science. 1991; 251: 675-678Crossref PubMed Scopus (1287) Google Scholar, 30Glenner GG Wong CW Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein.Biochem Biophys Res Commun. 1984; 120: 885-890Crossref PubMed Scopus (4433) Google Scholar, 31Lee VM Wang J Trojanowski JQ Purification of paired helical filament tau and normal tau from human brain tissue.Methods Enzymol. 1999; 309: 81-89Crossref PubMed Scopus (46) Google Scholar Subsequent genetic studies based on these discovered abnormal proteins ushered in the molecular era of investigation into neurodegenerative diseases.32Tsuang DW Bird TD Genetics of dementia.Med Clin North Am. 2002; 86: 591-614Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Given the apparent limitations of genomic screens to gain insight into the pathogenesis of PDC, here we pursued quantitative proteomics to discover those proteins resistant to surfactant extraction in PDC. Tissue from frontal cortex and temporal cortex was investigated from six different groups of individuals from two different parts of the world (Table 1). One set of tissue was from patients and controls in the Seattle area; all were Caucasian and had been followed clinically by the Alzheimer's Disease Research Center at the University of Washington. All patients from Seattle (AD, PSP, or dementia with Lewy bodies or DLB) had clinical and neuropathological diagnoses made according to established consensus criteria33Litvan I Agid Y Calne D Campbell G Dubois B Duvoisin RC Goetz CG Golbe LI Grafman J Growdon JH Hallett M Jankovic J Quinn NP Tolosa E Zee DS Clinical research criteria for the diagnosis of progressive supranuclear palsy (Steele-Richardson-Olszewski syndrome): report of the NINDS-SPSP international workshop.Neurology. 1996; 47: 1-9Crossref PubMed Scopus (2167) Google Scholar, 34McKeith I Galasko D Kosaka K Perry E Dickson D Hansen L Salmon D Lowe J Mirra S Byrne E Lennox G Quinn N Edwardson J Ince P Bergeron C Burns A Miller B Lovestone S Collerton D Jansen E Ballard C de Vos R Wilcock G Jellinger K Perry R Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the Consortium on DLB International Workshop.Neurology. 1996; 47: 1113-1124Crossref PubMed Scopus (3684) Google Scholar, 35The National Institute on Aging and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer's Disease Consensus recommendations for the postmortem diagnosis of Alzheimer's disease.Neurobiol Aging. 1997; 18: S1-S2Abstract Full Text Full Text PDF PubMed Scopus (1114) Google Scholar; patients with AD were selected to be free of coexisting Lewy body disease or vascular damage, whereas all patients with DLB had coexisting AD. AD cases with long postmortem interval (PMI) were selected to match PMI for individuals from Guam. All controls had been seen by the center physicians within 2 years of death and had normal neurological examinations, and all psychometric tests were within normal ranges. The second set of tissue was from controls and patients with PDC from Guam; all were Chamorro and had been followed clinically by the National Institutes of Health-funded research consortium and were diagnosed either as having PDC or as neurologically normal.Table 1Characteristics and Neuropathological Data of People Whose Tissue Was Used in This StudyCSADAD-LPMIDLBPSPCGPDCn6735367EthnicityAll Caucasian from Seattle AreaAll Chamorro from GuamWomen (n)4612124Age (years)70 ± 1584 ± 983 ± 686 ± 872 ± 1066 ± 1677 ± 10PMI (minutes)*P < 0.0001 by analysis of variance for all six groups.421 ± 186254 ± 722160 ± 720285 ± 91353 ± 701589 ± 6241539 ± 797CERAD neuritic plaque scoreNone (none to sparse)Frequent (moderate to frequent)Frequent (moderate to frequent)Frequent (moderate to frequent)NoneNone (none to sparse)None (none to sparse)Braak stageI (0 to III)VI (V or VI)VI (V or VI)V (V or VI)NA0 (0 to III)NA†Although Braak staging is not applicable (NA) to PDC, entorhinal cortex from all PDCs but no CGs had stage 3 or 4 NFT density (>20 NFTs/mm2), whereas frontal cortex from all PDCs had stage 1 or 2 NFT density (1 to 20 NFTs/mm2) and no CGs had any neocortical NFTs.37Neocortical LBs or Lewy neuritesNoneNoneNoneFive of fiveNoneNoneNone‡Two patients with PDC had Lewy bodies in the amygdala. Bonferroni-corrected post tests showed that all Seattle area groups had significantly shorter PMIs than groups from Guam (P < 0.01), except for AD-LPMI, whose PMI was not significantly different from CG or PDC.Continuous data are presented as mean ± SD, whereas discontinuous data are presented as mode (range).* P < 0.0001 by analysis of variance for all six groups.† Although Braak staging is not applicable (NA) to PDC, entorhinal cortex from all PDCs but no CGs had stage 3 or 4 NFT density (>20 NFTs/mm2), whereas frontal cortex from all PDCs had stage 1 or 2 NFT density (1 to 20 NFTs/mm2) and no CGs had any neocortical NFTs.37Perl DP Hof PR Purohit DP Loerzel AJ Kakulas BA Hippocampal and entorhinal cortex neurofibrillary tangle formation in Guamanian Chamorros free of overt neurologic dysfunction.J Neuropathol Exp Neurol. 2003; 62: 381-388PubMed Google Scholar‡ Two patients with PDC had Lewy bodies in the amygdala. Bonferroni-corrected post tests showed that all Seattle area groups had significantly shorter PMIs than groups from Guam (P < 0.01), except for AD-LPMI, whose PMI was not significantly different from CG or PDC. Open table in a new tab Continuous data are presented as mean ± SD, whereas discontinuous data are presented as mode (range). Braak system for staging NFT pathology in AD,36Braak H Braak E Neuropathological stageing of Alzheimer-related changes.Acta Neuropathol (Berl). 1991; 82: 239-259Crossref PubMed Scopus (12201) Google Scholar regional NFT density for characterizing PDC,37Perl DP Hof PR Purohit DP Loerzel AJ Kakulas BA Hippocampal and entorhinal cortex neurofibrillary tangle formation in Guamanian Chamorros free of overt neurologic dysfunction.J Neuropathol Exp Neurol. 2003; 62: 381-388PubMed Google Scholar and CERAD plaque score38Mirra S Heyman A McKeel D Sumi S Crain B Brownlee L Vogel F Hughes J van Belle G Berg L The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease.Neurology. 1991; 41: 479-486Crossref PubMed Google Scholar were accomplished with modified Bielschowsky-stained sections. All 37 cases underwent assessment for Lewy bodies and Lewy neurites in a single batch using SNCA immunohistochemistry (IHC) with antibody LB509 (1:50 to 1:400; Zymed, South San Francisco, CA).39Jakes R Crowther RA Lee VM Trojanowski JQ Iwatsubo T Goedert M Epitope mapping of LB509, a monoclonal antibody directed against human α-synuclein.Neurosci Lett. 1999; 269: 13-16Crossref PubMed Scopus (91) Google Scholar IHC for SNCA was performed on 10-μm sections that were pretreated with either 88% formic acid for 5 minutes or protease K for 1 minute, exposed to 3% hydrogen peroxide, blocked in 5% milk, incubated with primary antibody for 1 hour at room temperature, and then detected with avidin-biotin complex using diaminobenzidine as chromogen substrate.40Leverenz JB Fishel MA Peskind ER Montine TJ Nochlin D Steinbart E Raskind MA Schellenberg GD Bird TD Tsuang D Lewy body pathology in familial Alzheimer disease—evidence for disease- and mutation-specific pathologic phenotype.Arch Neurol. 2006; 63: 370-376Crossref PubMed Scopus (106) Google Scholar The positive control for each IHC run was a case of DLB. Negative control for each was elimination of primary antibody. Tissue was homogenized and sequentially extracted in buffer A [10 mmol/L Tris, 1 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1 mmol/L dithiothreitol, and 10% sucrose, pH 7.5], buffer B (buffer A + 1% Triton X-100), and buffer C (buffer A + 1% N-lauroylsarcosine). Phosphatase inhibitors (20 mmol/L NaF and 1 mmol/L sodium orthovanadate) and protease inhibitor cocktail (no. P2714; Sigma Chemical Co., St. Louis, MO) were added to buffers A, B, and C immediately before use. Surfactant-insoluble material insoluble in buffer B (Triton-insoluble or TI) or buffer C (sarkosyl-insoluble or SI) was extracted with 70% formic acid as previously described.41Woltjer RL Cimino PJ Boutte AM Schantz AM Montine KS Larson EB Bird T Quinn JF Zhang J Montine TJ Proteomic determination of widespread detergent-insolubility including Aβ but not tau early in the pathogenesis of Alzheimer's disease.FASEB J. 2005; 19: 1923-1925PubMed Google Scholar Formic acid extracts of surfactant-insoluble proteins were dried by vacuum centrifugation and resolubilized by sonication in 20 vol of 5 mol/L guanidine HCl, 100 mmol/L Tris, pH 7.4, with 0.002% bromphenol blue added to confirm elimination of formic acid. These were further diluted 64-fold in 100 mmol/L Tris, pH 7.4, with 0.05% azide and 0.002% bromphenol blue, and 100 μl (representing 7.8 μg of starting brain tissue) were spotted onto 96-well plates and then incubated overnight at room temperature in a humidified chamber. Plates were subsequently washed twice with phosphate-buffered saline (PBS), blocked with 1% bovine serum albumin in PBS with 0.05% sodium azide, and washed again with PBS. ELISAs were performed as described by others and developed using tetramethylbenzidine with absorbances determined at 405 nm.42Harlow E Lane D Antibodies.A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1988Google Scholar Primary antibodies were directed against Aβ40 and Aβ42 (carboxy-terminal-specific antibodies; Signet Laboratories, Dedham, MA), tau, apoE, ubiquitin (all from DAKO, Carpinteria, CA), and SCNA (from Chemicon, Norcross, GA; or LabVision Neomarkers, Fremont, CA). Antibodies specific to tau phosphorylated at Ser199 and Ser396 were purchased from Invitrogen (Carlsbad, CA). Antibody specific to PHF tau phosphorylated at Ser212/Thr214 (AT100) was obtained from Pierce (Rockford, IL). Antibody specificity was confirmed by Western blot of brain extracts before use in capture assays. Secondary antibody/alkaline phosphatase conjugates were from Amersham (Piscataway, NJ). All antibodies were diluted 2000 times from the initial stock concentration before use in the detection assay. Luminex assays for soluble Aβ oligomers were performed on material solubilized by buffer A and buffer B using reagents from Biosource (Camarillo, CA) exactly according to the manufacturer's instructions. This paired fluorescent bead-antibody conjugate assay uses a bead-A11 antibody conjugate that detects oligomers, but not monomers or fibrils, of several proteins that form amyloid including Aβ species,43Kayed R Sokolov Y Edmonds B McIntire TM Milton SC Hall JE Glabe CG Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases.J Biol Chem. 2004; 279: 46363-46366Crossref PubMed Scopus (789) Google Scholar paired with a second bead-antibody conjugate that detects an epitope within the first 20 amino acids of the Aβ peptide. Standards were provided by the manufacturer, and standard curves were generated throughout 30 to 2000 pg/ml Aβ42 oligomers with a detection limit of 30 pg/ml. SI protein was solubilized by sonication in 8 mol/L urea with 500 mmol/L triethylamine bicarbonate, pH 8.5, and equal amounts of SI protein was pooled for each group to yield 100 μg of SI protein from patients with PDC, AD, controls from Guam (CG), and controls from Seattle (CS); these were digested in parallel with trypsin and then labeled with one of the four-iTRAQ reagents following the manufacturer's instructions. The four iTRAQ-labeled samples were combined (a total of 400 μg of proteins) and loaded onto a strong cation exchange column (0.5 × 200 mm) that had been equilibrated in 0.05% formic acid/20% acetonitrile (ACN), and pH 3.0, at a flow rate of 200 μl/minute. Peptides were eluted by applying a linear gradient from 0 to 100% of 500 mmol/L ammonium formiate/20% ACN, pH 3.0. Eleven fractions were collected from each sample and dried down in a SpeedVac (Thermo Savant, Holbrook, NY). Strong cation exchange-fractionated peptides were then dissolved in 0.5% trifluoroacetic acid and separated using reverse phase chromatography. Nano-capillary liquid chromatography (LC) was performed using the UltiMate with Famos autosampler and Switchos automated switching valve (LC Packings, Sunnyvale, CA). Samples were loaded onto a capillary precolumn cartridge (Dionex, Sunnyvale, CA). The trap column was washed with mobile phase A containing 2% ACN and 0.1% trifluoroacetic acid in high-performance liquid chromatography water. The flow rate was set at 0.4 μl/minute. The sample was then loaded onto a 15 cm × 100-μm ID Magic C18 (3 μm), 100-angstrom packing capillary liquid chromatography column (Michrome BioResources Inc., Auburn, CA). The gradient run was from 5 to 90% mobile phase B (80% ACN, 20% high-performance liquid chromatography water, and 0.08% trifluoroacetic acid) for 85 minutes. The eluate was mixed with 7 mg/ml of recrystallized α-cyano-4-hydroxycinnamic acid (Sigma) in 60% ACN, 2.6% (5 mg/ml) ammonium citrate with internal standard (4700 mass standard kit; Applied Biosystems, Foster City, CA), and spotted onto a stainless steel matrix-assisted laser desorption ionization plate with the Probot (LC Packings). Samples were spotted at 5-second intervals using a 24 × 24 array pattern for a total of 576 spots per plate. In total, 11 liquid chromatography matrix-assisted laser desorption ionization plates were spotted and analyzed by a 4700 proteomic system (see below; Applied Biosystems). Quantitative mass spectrometry (MS) analysis was performed using the 4700 proteomics analyzer with time-of-flight (TOF)/TOF optics (Applied Biosystems). MS reflector-positive ion mode with automated acquisition of 800 to 4000 m/z range was used with 1000 shots per spectrum. A maximum of 15 peaks was selected per spot, with a minimum signal-noise (S/N) ratio of 75 and cluster area of 500. Greater than 36,000 precursors were selected and were submitted for tandem mass spectrometry in positive ion mode with CID cell on and 1 kV collision energy; 3000 shots were accumulated per spectrum. For each spotted plate, a total of 576 MS and more than 1200 tandem mass spectrometry spectra were acquired. Identification of proteins was achieved using Mascot (Matrix Science, Boston, MA) algorithm and searched against the International Protein Index Version 3.18 (http://www.ebi.ac.uk/IPI). Criteria for all protein identification were at least one peptide with individual composite score greater than the 95% confidence interval threshold and also the top-ranked matching sequence for that spectrum. For proteins identified by a single peptide, two additional criteria were applied: the Mascot ion
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