Abnormal Neurites Containing C-Terminally Truncated α-Synuclein Are Present in Alzheimer's Disease without Conventional Lewy Body Pathology
2010; Elsevier BV; Volume: 177; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2010.100552
ISSN1525-2191
AutoresKaren A. Lewis, Yang Su, Olina Jou, Caroline Ritchie, Chan Foong, Linda S. Hynan, Charles L. White, Philip Thomas, Kimmo J. Hatanpaa,
Tópico(s)Alzheimer's disease research and treatments
ResumoThe pathological hallmark of Parkinson's disease and diffuse Lewy body disease (DLBD) is the aggregation of α-synuclein (α-syn) in the form of Lewy bodies and Lewy neurites. Patients with both Alzheimer's disease (AD) and cortical Lewy pathology represent the Lewy body variant of AD (LBV) and constitute 25% of AD cases. C-terminally truncated forms of α-syn enhance the aggregation of α-syn in vitro. To investigate the presence of C-terminally truncated α-syn in DLBD, AD, and LBV, we generated and validated polyclonal antibodies to truncated α-syn ending at residues 110 (α-syn110) and 119 (α-syn119), two products of 20S proteosome-mediated endoproteolytic cleavage. Double immunofluorescence staining of the cingulate cortex showed that α-syn110 and α-syn140 (full-length) aggregates were not colocalized in LBV. All aggregates containing α-syn140 also contained α-syn119; however, some aggregates contained α-syn119 without α-syn140, suggesting that α-syn119 may stimulate aggregate formation. Immunohistochemistry and image analysis of tissue microarrays of the cingulate cortex from patients with DLBD (n = 27), LBV (n = 27), and AD (n = 19) and age-matched controls (n = 15) revealed that AD is also characterized by frequent abnormal neurites containing α-syn119. Notably, these neurites did not contain α-syn ending at residues 110 or 122-140. The presence of abnormal neurites containing α-syn119 in AD without conventional Lewy pathology suggests that AD and Lewy body disease may be more closely related than previously thought. The pathological hallmark of Parkinson's disease and diffuse Lewy body disease (DLBD) is the aggregation of α-synuclein (α-syn) in the form of Lewy bodies and Lewy neurites. Patients with both Alzheimer's disease (AD) and cortical Lewy pathology represent the Lewy body variant of AD (LBV) and constitute 25% of AD cases. C-terminally truncated forms of α-syn enhance the aggregation of α-syn in vitro. To investigate the presence of C-terminally truncated α-syn in DLBD, AD, and LBV, we generated and validated polyclonal antibodies to truncated α-syn ending at residues 110 (α-syn110) and 119 (α-syn119), two products of 20S proteosome-mediated endoproteolytic cleavage. Double immunofluorescence staining of the cingulate cortex showed that α-syn110 and α-syn140 (full-length) aggregates were not colocalized in LBV. All aggregates containing α-syn140 also contained α-syn119; however, some aggregates contained α-syn119 without α-syn140, suggesting that α-syn119 may stimulate aggregate formation. Immunohistochemistry and image analysis of tissue microarrays of the cingulate cortex from patients with DLBD (n = 27), LBV (n = 27), and AD (n = 19) and age-matched controls (n = 15) revealed that AD is also characterized by frequent abnormal neurites containing α-syn119. Notably, these neurites did not contain α-syn ending at residues 110 or 122-140. The presence of abnormal neurites containing α-syn119 in AD without conventional Lewy pathology suggests that AD and Lewy body disease may be more closely related than previously thought. The deposition of aggregated α-synuclein (α-syn) is the pathological hallmark of Lewy body disease, which includes Parkinson's disease (PD), diffuse Lewy body disease (DLBD), and Lewy body variant of Alzheimer's disease (LBV) (Figure 1). By immunohistochemistry, the aggregated α-syn in Lewy body disease is localized to round cytoplasmic inclusions known as Lewy bodies and to abnormal neurites known as Lewy neurites. Some of the aggregated α-syn in human brain tissue is C-terminally truncated, a modification that has been shown to accelerate oligomer and amyloid formation in vitro.1Hoyer W Cherny D Subramaniam V Jovin TM Impact of the acidic C-terminal region comprising amino acids 109-140 on alpha-synuclein aggregation in vitro.Biochemistry. 2004; 43: 16233-16242Crossref PubMed Scopus (274) Google Scholar, 2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar, 3Li W West N Colla E Pletnikova O Troncoso JC Marsh L Dawson TM Jakala P Hartmann T Price DL Lee MK Aggregation promoting C-terminal truncation of alpha-synuclein is a normal cellular process and is enhanced by the familial Parkinson's disease-linked mutations.Proc Natl Acad Sci USA. 2005; 102: 2162-2167Crossref PubMed Scopus (367) Google Scholar Furthermore, substochiometric amounts of truncated forms can seed the aggregation of the full-length protein,2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar which leads to the attractive hypothesis that truncated α-syn contributes to early stages of pathogenesis. Consistent with this hypothesis, production of truncated forms of α-syn correlate with disease and cause PD-like phenotypes in transgenic animals. Truncations of α-syn are present in the insoluble, fibrillary aggregates found in the brains of Lewy body disease patients, but not in aggregate fractions of control brains.2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar, 4Anderson JP Walker DE Goldstein JM de Laat R Banducci K Caccavello RJ Barbour R Huang J Kling K Lee M Diep L Keim PS Shen X Chataway T Schlossmacher MG Seubert P Schenk D Sinha S Gai WP Chilcote TJ Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease.J Biol Chem. 2006; 281: 29739-29752Crossref PubMed Scopus (897) Google Scholar Appearance of truncated α-syn also correlates with pathogenesis in transgenic mouse models of PD.2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar, 3Li W West N Colla E Pletnikova O Troncoso JC Marsh L Dawson TM Jakala P Hartmann T Price DL Lee MK Aggregation promoting C-terminal truncation of alpha-synuclein is a normal cellular process and is enhanced by the familial Parkinson's disease-linked mutations.Proc Natl Acad Sci USA. 2005; 102: 2162-2167Crossref PubMed Scopus (367) Google Scholar, 5Daher JP Ying M Banerjee R McDonald RS Hahn MD Yang L Flint Beal M Thomas B Dawson VL Dawson TM Moore DJ Conditional transgenic mice expressing C-terminally truncated human alpha-synuclein (alphaSyn119) exhibit reduced striatal dopamine without loss of nigrostriatal pathway dopaminergic neurons.Mol Neurodegener. 2009; 4: 34Crossref PubMed Scopus (70) Google Scholar Finally, transgenic mice that express C-terminally truncated forms of human α-syn develop PD-like symptoms and exhibit PD-like neuronal pathology.6Wakamatsu M Ishii A Iwata S Sakagami J Ukai Y Ono M Kanbe D Muramatsu SI Kobayashi K Iwatsubo T Yoshimoto M Selective loss of nigral dopamine neurons induced by overexpression of truncated human alpha-synuclein in mice.Neurobiol Aging. 2008; 29: 574-585Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 7Tofaris GK Garcia Reitbock P Humby T Lambourne SL O'Connell M Ghetti B Gossage H Emson PC Wilkinson LS Goedert M Spillantini MG Pathological changes in dopaminergic nerve cells of the substantia nigra and olfactory bulb in mice transgenic for truncated human alpha-synuclein(1-120): implications for Lewy body disorders.J Neurosci. 2006; 26: 3942-3950Crossref PubMed Scopus (260) Google Scholar The mechanism that produces truncated α-syn in vivo has not been conclusively identified. Multiple proteases have been shown to cleave α-syn, including the 20S proteasome,2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar, 8Bennett MC Bishop JF Leng Y Chock PB Chase TN Mouradian MM Degradation of alpha-synuclein by proteasome.J Biol Chem. 1999; 274: 33855-33858Crossref PubMed Scopus (388) Google Scholar calpain I,9Mishizen-Eberz AJ Guttmann RP Giasson BI Day 3rd, GA Hodara R Ischiropoulos H Lee VM Trojanowski JQ Lynch DR Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro.J Neurochem. 2003; 86: 836-847Crossref PubMed Scopus (129) Google Scholar, 10Mishizen-Eberz AJ Norris EH Giasson BI Hodara R Ischiropoulos H Lee VM Trojanowski JQ Lynch DR Cleavage of alpha-synuclein by calpain: potential role in degradation of fibrillized and nitrated species of alpha-synuclein.Biochemistry. 2005; 44: 7818-7829Crossref PubMed Scopus (98) Google Scholar and cathepsin D.11Sevlever D Jiang P Yen SH Cathepsin D is the main lysosomal enzyme involved in the degradation of alpha-synuclein and generation of its carboxy-terminally truncated species.Biochemistry. 2008; 47: 9678-9687Crossref PubMed Scopus (216) Google Scholar Previous work in one of our laboratories (P.J.T.) showed that abnormally aggregated α-syn contains at least three truncated α-syn forms in addition to full-length α-syn.2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar Although the exact peptide sequence of the three truncated α-syn forms was unclear, epitope mapping indicated that two of these proteins were C-terminally truncated, ending at approximately amino acid residues 102-125 and 83-110, respectively. These two C-terminally truncated proteins had intact N-termini and were consistent with the 1-119, 1-122, and the 1-110 truncations produced by the 20S proteasome in vitro.2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar The third truncated α-syn form was both C- and N-terminally truncated and appeared to consist mainly of the central, highly amyloidogenic NAC (non-Aβ component) fragment of α-syn,2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar which corresponds to amino acid residues 61-95. The NAC fragment is not associated with the Aβ peptide in AD plaques as originally thought, but it has been suggested that NAC may be present within dystrophic, phospho-τ-immunoreactive neurites surrounding neuritic AD plaques.12Culvenor JG McLean CA Cutt S Campbell BC Maher F Jakala P Hartmann T Beyreuther K Masters CL Li QX Non-Abeta component of Alzheimer's disease amyloid (NAC) revisited. NAC and alpha-synuclein are not associated with Abeta amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar Another study, based on mass spectrometry after trypsin digestion, indicated the presence of C-terminally truncated α-syn ending at amino acids 119, 122, and possibly 123 in the human brain.3Li W West N Colla E Pletnikova O Troncoso JC Marsh L Dawson TM Jakala P Hartmann T Price DL Lee MK Aggregation promoting C-terminal truncation of alpha-synuclein is a normal cellular process and is enhanced by the familial Parkinson's disease-linked mutations.Proc Natl Acad Sci USA. 2005; 102: 2162-2167Crossref PubMed Scopus (367) Google Scholar In addition, shorter fragments are present but could not be analyzed due to their low abundance. The C-terminally truncated forms of α-syn were seen in the particulate fractions from Lewy body disease brains but also in the soluble fractions of age-similar control brains and cultured cells.2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar, 3Li W West N Colla E Pletnikova O Troncoso JC Marsh L Dawson TM Jakala P Hartmann T Price DL Lee MK Aggregation promoting C-terminal truncation of alpha-synuclein is a normal cellular process and is enhanced by the familial Parkinson's disease-linked mutations.Proc Natl Acad Sci USA. 2005; 102: 2162-2167Crossref PubMed Scopus (367) Google Scholar The 20S core particle of the proteasome is capable of endoproteolytically cleaving α-syn in vitro in a ubiquitin-independent manner,13Liu CW Corboy MJ DeMartino GN Thomas PJ Endoproteolytic activity of the proteasome.Science. 2003; 299: 408-411Crossref PubMed Scopus (347) Google Scholar producing fragments that are similar to those observed in patient samples and that promote the formation of putative cytotoxic oligomer species.2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar, 3Li W West N Colla E Pletnikova O Troncoso JC Marsh L Dawson TM Jakala P Hartmann T Price DL Lee MK Aggregation promoting C-terminal truncation of alpha-synuclein is a normal cellular process and is enhanced by the familial Parkinson's disease-linked mutations.Proc Natl Acad Sci USA. 2005; 102: 2162-2167Crossref PubMed Scopus (367) Google Scholar, 14Lewis KA Yaeger A DeMartino GN Thomas PJ Accelerated formation of alpha-synuclein oligomers by concerted action of the 20S proteasome and familial Parkinson mutations.J Bioenerg Biomembr. 2010; 42: 85-95Crossref PubMed Scopus (18) Google Scholar In addition to its central role in the Lewy body diseases, α-syn pathology, excluding amygdala-only pathology, has been observed in 34% of patients with significant concomitant Alzheimer's disease (AD) pathology.15Uchikado H Lin WL DeLucia MW Dickson DW Alzheimer disease with amygdala Lewy bodies: a distinct form of alpha-synucleinopathy.J Neuropathol Exp Neurol. 2006; 65: 685-697Crossref PubMed Scopus (256) Google Scholar The coexistence of α-syn and AD pathology is known as Lewy body variant of AD (LBV) (Figure 1). In cases with concurrent AD pathology, consisting of aggregated Aβ peptide and abnormally phosphorylated tau (phospho-tau), α-syn pathology is generally much more severe than in cases without AD pathology.16Pletnikova O West N Lee MK Rudow GL Skolasky RL Dawson TM Marsh L Troncoso JC Abeta deposition is associated with enhanced cortical alpha-synuclein lesions in Lewy body diseases.Neurobiol Aging. 2005; 26: 1183-1192Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar Interactions that stimulate cross-fibrillization have been identified in vitro between α-syn and Aβ peptide as well as between α-syn and phospho-tau.17Giasson BI Forman MS Higuchi M Golbe LI Graves CL Kotzbauer PT Trojanowski JQ Lee VM Initiation and synergistic fibrillization of tau and alpha-synuclein.Science. 2003; 300: 636-640Crossref PubMed Scopus (709) Google Scholar, 18Geddes JW alpha-Synuclein: a potent inducer of tau pathology.Exp Neurol. 2005; 192: 244-250Crossref PubMed Scopus (43) Google Scholar Interactions are also suggested at the genetic level, as a specific haplotype of the tau gene increases the risk for Parkinson's disease.19Mamah CE Lesnick TG Lincoln SJ Strain KJ de Andrade M Bower JH Ahlskog JE Rocca WA Farrer MJ Maraganore DM Interaction of alpha-synuclein and tau genotypes in Parkinson's disease.Ann Neurol. 2005; 57: 439-443Crossref PubMed Scopus (46) Google Scholar In addition to the increased full-length α-syn pathology in LBV, truncated forms of α-syn also appear to be more abundant in cases with concurrent AD pathology.16Pletnikova O West N Lee MK Rudow GL Skolasky RL Dawson TM Marsh L Troncoso JC Abeta deposition is associated with enhanced cortical alpha-synuclein lesions in Lewy body diseases.Neurobiol Aging. 2005; 26: 1183-1192Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar Because truncations of α-syn are known to increase amyloidogenicity and a strong possibility for cross-seeding of amyloidogenic proteins exists, the identification of specific truncated α-syn forms may contribute to the understanding of the pathogenesis of not only Lewy body disease but also LBV and AD. Furthermore, the presence of specific truncations may also contribute to the understanding of the complex boundaries between these diseases. Ultimately, accurate clinical distinction between Lewy body disease and AD has direct consequences for treatment, as some medications are contra-indicated for Lewy body disease that are acceptable for use by AD patients.20McKeith I Fairbairn A Perry R Thompson P Perry E Neuroleptic sensitivity in patients with senile dementia of Lewy body type.Bmj. 1992; 305: 673-678Crossref PubMed Scopus (551) Google Scholar Antibodies that specifically recognize truncated forms of α-syn would be useful for high-throughput analyses of both cultured cells and tissues. An approach for the development of antibodies that incorporate the C-terminal carboxylic acid group into the recognition epitope has been previously described.21Liang TC Luo W Hsieh JT Lin SH Antibody binding to a peptide but not the whole protein by recognition of the C-terminal carboxy group.Arch Biochem Biophys. 1996; 329: 208-214Crossref PubMed Scopus (14) Google Scholar In this study, polyclonal antibodies were raised using a similar approach against two forms of truncated α-syn: α-syn ending at amino acids 110 (α-syn110) and 119 (α-syn119). The antibodies, named syn110 and syn119, specifically recognized the corresponding truncated forms of α-syn as evaluated by Western blotting. Both new antibodies detected α-syn pathological aggregates in Lewy body disease, but only α-syn119 aggregates were found to be predominantly colocalized with full-length α-syn. Abnormal neurites containing α-syn119 were detected in AD in the absence of other forms of pathological α-syn. The peptide antigens corresponded to the last seven amino acids of α-syn110 and α-syn119, respectively (Table 1). Protein BLAST (http://blast.ncbi.nlm.nih.gov, last accessed October 16, 2010.) searches showed no significant homology between the peptides and human proteins other than α-syn. To ensure that the C terminus would be available for incorporation into the recognition epitope, an N-terminal cysteine residue was added to the peptide sequence (Table 1). The peptides were then conjugated to the commonly used carrier protein keyhole limpet hemocyanin (KLH) via maleimide chemistry. Immunization of rabbits was contracted to Proteintech Group (Chicago, IL). For each antigen, two rabbits were immunized and preimmune and final bleed sera were collected from each animal. The antisera were affinity purified using columns with immobilized antigenic peptides. The purified antibodies, named syn110 and syn119, were aliquoted and stored at −20°C in 0.02% NaN3.Table 1Synuclein AntibodiesAntibody designationAntigen (peptide sequence)/epitopeTypeClone (catalog number)SourceSyn110α-syn aa. 104-110 (N′-CEEGAPQE*To ensure that the C-terminus would be available for incorporation into the epitope, an N-terminal cysteine residue was added to the peptide sequence.)Rabbit polyclonalN/AThis studySyn119α-syn aa. 113-119 (N′-CLEDMPVD*To ensure that the C-terminus would be available for incorporation into the epitope, an N-terminal cysteine residue was added to the peptide sequence.)Rabbit polyclonalN/AThis studySyn122-140α-syn aa. 115-122†Amino acid 122 is required for recognition.22Mouse monoclonalLB509InvitrogenSyn140C-terminus of α-syn (aa. 130-140) and β-synMouse monoclonalSyn 202AbcamN-ter synN-terminus of α-syn and β-synRabbit polyclonalGTX15534GeneTexPan-synucleinα-syn aa. 83-100‡Reference.2Mouse monoclonal42/α-Synuclein (610786)BD Transduction Laboratoriesaa., amino acids.* To ensure that the C-terminus would be available for incorporation into the epitope, an N-terminal cysteine residue was added to the peptide sequence.† Amino acid 122 is required for recognition.22Jakes R Crowther RA Lee VM Trojanowski JQ Iwatsubo T Goedert M Epitope mapping of LB509, a monoclonal antibody directed against human alpha-synuclein.Neurosci Lett. 1999; 269: 13-16Crossref PubMed Scopus (90) Google Scholar‡ Reference.2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar Open table in a new tab aa., amino acids. The generated polyclonal antibodies were validated against the intended targets using recombinantly expressed and purified truncations of α-syn in an immunoblot assay (Figure 2). Recombinant α-syn proteins were expressed and purified as described.2Liu CW Giasson BI Lewis KA Lee VM Demartino GN Thomas PJ A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.J Biol Chem. 2005; 280: 22670-22678Crossref PubMed Scopus (214) Google Scholar Because these truncated proteins have a tendency to aggregate during storage, solutions of the recombinant proteins were filtered to reduce the amount of protein aggregates immediately before loading the gel. A 30-μL aliquot of each recombinant protein was applied on a Microcon YM-100 filter column (Millipore, Billerica, MA) and centrifuged at 16,000 × g for 10 minutes at 4°C. Of the flow-through, 10 μl was dissolved in 30 μl of 8M guanidine hydrochloride (final concentration, 6M) and used for determination of protein concentration with the Micro BCA protein assay kit (Thermo Scientific, Rockford, IL), while the remaining flow-through was used for immunoblotting. Each recombinant protein, 200 ng/lane, was suspended in NuPage LDS Sample Buffer (Invitrogen, Carlsbad, CA) with 50 mmol/L dithiothreitol, heated at 70°C for 10 minutes, and loaded on NuPage 10% Bis-Tris gel. After electrophoresis and transfer to nitrocellulose, the membranes were exposed to the syn110 or syn119 antibody diluted 1:2000 in Tris-buffered saline with 1% milk and 0.1% Tween-20 for 1 hour at RT and then to peroxidase-conjugated goat anti-rabbit (H+L) (#32460, Thermo) diluted 1:3000. The signal was detected using SuperSignal West Dura Extended Duration Substrate (Thermo) and CL-XPosure film (Thermo). The membrane was then stripped using Restore Western blot Stripping Buffer (Thermo) and reprobed with the pan-synuclein antibody (Table 1) diluted 1:2000. Samples of human brain tissue were obtained from the Neuropathology Core of the Alzheimer's Disease Center at University of Texas Southwestern Medical Center (UT Southwestern). At autopsy, one of the cerebral hemispheres was sectioned coronally at 1-cm intervals. The slabs were placed in plastic bags and stored at −80°C. The other hemisphere was fixed in formalin and sampled for paraffin sections. Approximately 0.5 grams of frozen cortical tissue was dissected from the anterior cingulate gyrus and used for biochemical studies. A signed consent for brain autopsy had been obtained from the next of kin or legal representative in each case. The Institutional Review Board (IRB) of UT Southwestern, based on federal regulations (45CFR 46), has determined that studies using autopsy tissue are not considered "human research" and therefore no IRB approval is required. Brain autopsy cases of AD, DLBD, LBV, and controls were selected for this study based on availability. The diagnosis of AD was based on densities of neuritic plaques, detected using a modified thioflavin S stain,23Guntern R Bouras C Hof PR Vallet PG An improved thioflavine S method for staining neurofibrillary tangles and senile plaques in Alzheimer's disease.Experientia. 1992; 48: 8-10Crossref PubMed Scopus (113) Google Scholar meeting the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) criteria for "definite AD".24Mirra SS Hart MN Terry RD Making the diagnosis of Alzheimer's disease. A primer for practicing pathologists.Arch Pathol Lab Med. 1993; 117: 132-144PubMed Google Scholar Seventeen of the 19 AD cases selected in this manner had a Braak neurofibrillary tangle stage25Braak H Alafuzoff I Arzberger T Kretzschmar H Del Tredici K Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry.Acta Neuropathol. 2006; 112: 389-404Crossref PubMed Scopus (1828) Google Scholar of at least V, and the remaining two cases had a Braak stage of IV. The diagnosis of DLBD (n = 27) was based on meeting the criteria for "neocortical stage" of Lewy body disease according to the Third Report of the Dementia with Lewy bodies Consortium (Third Consortium),26McKeith IG Dickson DW Lowe J Emre M O'Brien JT Feldman H Cummings J Duda JE Lippa C Perry EK Aarsland D Arai H Ballard CG Boeve B Burn DJ Costa D Del Ser T Dubois B Galasko D Gauthier S Goetz CG Gomez-Tortosa E Halliday G Hansen LA Hardy J Iwatsubo T Kalaria RN Kaufer D Kenny RA Korczyn A Kosaka K Lee VM Lees A Litvan I Londos E Lopez OL Minoshima S Mizuno Y Molina JA Mukaetova-Ladinska EB Pasquier F Perry RH Schulz JB Trojanowski JQ Yamada M Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium.Neurology. 2005; 65: 1863-1872Crossref PubMed Scopus (4131) Google Scholar not meeting the CERAD neuritic plaque criteria for "definite AD,"24Mirra SS Hart MN Terry RD Making the diagnosis of Alzheimer's disease. A primer for practicing pathologists.Arch Pathol Lab Med. 1993; 117: 132-144PubMed Google Scholar and not having a Braak tangle stage higher than IV.25Braak H Alafuzoff I Arzberger T Kretzschmar H Del Tredici K Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry.Acta Neuropathol. 2006; 112: 389-404Crossref PubMed Scopus (1828) Google Scholar The diagnosis of LBV (n = 27) was based on meeting the Third Consortium "neocortical stage" criteria of Lewy body disease and also meeting the CERAD "definite" criteria for AD. The widely-used CERAD criteria were chosen because they emphasize neuritic plaques, a type of senile plaques, and previous evidence suggests that Aβ deposition in the form of senile plaques is associated with enhanced cortical α-syn lesions in Lewy body disease.16Pletnikova O West N Lee MK Rudow GL Skolasky RL Dawson TM Marsh L Troncoso JC Abeta deposition is associated with enhanced cortical alpha-synuclein lesions in Lewy body diseases.Neurobiol Aging. 2005; 26: 1183-1192Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar Age-similar controls (n = 15) had no clinical history of dementia or cognitive impairment, met the CERAD criteria for a normal control, had a CERAD age-related neuritic plaque score of at most "A," and had a Braak stage of at most II. The mean ages for the AD, DLBD, LBV, and control groups were 73, 78, 77, and 78 years, respectively; the differences were not statistically significant by analysis of variance. Six cases of Parkinson's disease (PD), defined as clinical parkinsonism without dementia and with α-syn pathology at autopsy,27Weisman D McKeith I Dementia with Lewy bodies.Semin Neurol. 2007; 27: 42-47Crossref PubMed Scopus (84) Google Scholar were identified based on availability and included in the study. The PD cases were obtained through the Dallas County Medical Examiner's Office. Because the average age in the PD group (62 years) was lower than that in the other groups and also because of the relatively low number of cases in this group, the PD group was analyzed only qualitatively and separately from the other groups. Visible white matter and leptomeninges were dissected from the cortical brain sample. The sample was weighed and suspended in 2% (for subsequent immunoblotting using the syn110 antibody) or 4% (for syn119 antibody) SDS (3 ml of buffer for 1 g of tissue) with 20 mmol/L NaCl, 20 mmol/L Tris (pH 7.6), and Complete Mini EDTA-free Protease Inhibitor Cocktail (one tablet/10 ml; Roche). The sample was dissolved by sonicating six times for 15 seconds while the tube was immersed in an ice bath, pausing for 15 seconds between the periods of sonication, using a Microson Ultrasonic Cell Disruptor (Misonic, Farmingdale, NY) at 50% duty cycle and power output level 2. The samples were centrifuged at 100,000 × g (78,000 rpm, 15–20 psi) for 30 minutes at RT using an A-100/18 rotor in an Airfuge ultracentrifuge (Beckman Coulter, Brea, CA). The supernatant was retained as the SDS fraction. The pellet was washed once with SDS buffer and centrifuged again at 100,000 × g for 30 minutes. The pellet was dissolved in 8 mol/L urea buffer (1.5 ml/g of original sample weight) with 4% SDS, 4% CHAPS, 20 mmol/L NaCl, and 20 mmol/L Tris (pH 7.6). The sample was sonicated once for 15 seconds at the same settings as stated above and centrifuged at 1
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