Disruption of Amyloid Plaques Integrity Affects the Soluble Oligomers Content from Alzheimer Disease Brains

RESEARCH ARTICLE Disruption of Amyloid Plaques Integrity Affects the Soluble Oligomers Content from Alzheimer Disease Brains Sebastian Jimenez 1,2,4. , Victoria Navarro 1,2,4. , Javier Moyano 1,2,4 , Marı´a Sanchez-Mico 1,2,4 , Manuel Torres 1,2¤ , Jose Carlos Davila 3,4 , Marisa Vizuete 1,2,4 , Antonia Gutierrez 3,4" *, Javier Vitorica 1,2,4" * 1. Departamento Bioquı´mica y Biologı´a Molecular, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain, 2. Instituto de Biomedicina de Sevilla (IBiS)-Hospital Universitario Virgen del Rocı´o/CSIC/Universidad de Sevilla, Sevilla, Spain, 3. Departamento Biologı´a Celular, Gene´tica y Fisiologı´a, Facultad de Ciencias, Instituto de Investigacio´n Biome´dica de Ma´laga (IBIMA), Universidad de Ma´laga, Ma´laga, Spain, 4. Centro de Investigacio´n Biome´dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain *[email protected] (JV);agutierr[email protected] (AG) .These authors contributed equally to this work. "AG and JV are co-senior authors on this work. ¤ Current address: Laboratory of Molecular Cell Biomedicine, University of Balearic Islands, Palma, Spain Abstract The implication of soluble Abeta in the Alzheimer’s disease (AD) pathology is currently accepted. In fact, the content of soluble extracellular Abeta species, such as monomeric and/or oligomeric Abeta, seems to correlate with the clinicopathological dysfunction observed in AD patients. However, the nature (monomeric, dimeric or other oligomers), the relative abundance, and the origin (extra-/ intraneuronal or plaque-associated), of these soluble species are actually under debate. In this work we have characterized the soluble (defined as soluble in Trisbuffered saline after ultracentrifugation) Abeta, obtained from hippocampal samples of Braak II, Braak III–IV and Braak V–VI patients. Although the content of both Abeta40 and Abeta42 peptides displayed significant increase with pathology progression, our results demonstrated the presence of low, pg/mg protein, amount of both peptides. This low content could explain the absence (or below detection limits) of soluble Abeta peptides detected by western blots or by immunoprecipitation-western blot analysis. These data were in clear contrast to those published recently by different groups. Aiming to explain the reasons that determine these substantial differences, we also investigated whether the initial homogenization could mobilize Abeta from plaques, using 12-month-old PS1xAPP cortical samples. Our data demonstrated that manual homogenization (using Dounce) preserved the integrity of Abeta plaques whereas strong homogenization OPEN ACCESS Citation: Jimenez S, Navarro V, Moyano J, Sanchez-Mico M, Torres M, et al. (2014) Disruption of Amyloid Plaques Integrity Affects the Soluble Oligomers Content from Alzheimer Disease Brains. PLoS ONE 9(12): e114041. doi:10.1371/ journal.pone.0114041 Editor: Ramon Trullas, IIBB/CSIC/IDIBAPS, Spain Received: July 31, 2014 Accepted: November 3, 2014 Published: December 8, 2014 Copyright: ß2014 Jimenez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This work was supported by Fondo de Investigacio´n Sanitaria (FIS), from Instituto de Salud Carlos III of Spain, through grants PI12/ 01431 (to AG) and PI12/01439 (to JV), by Junta de Andalucia (collaborative grant CTS-2035 to JV and AG), and by Centro de Investigacion Biomedica en Red Sobre Enfermedades Neurodegenerativas (CIBERNED) (collaborative grants PI2010/08 and PI2013/01 to JV and AG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors confirm that the co-author Javier Vitorica is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria. PLOS ONE | DOI:10.1371/journal.pone.0114041 December 8, 2014 1/17 procedures (such as sonication) produced a vast redistribution of the Abeta species in all soluble and insoluble fractions. This artifact could explain the dissimilar and somehow controversial data between different groups analyzing human AD samples. Introduction Progressive aggregation and accumulation of extracellular amyloid-b(Abeta) peptides is central to Alzheimer’s disease (AD) pathogenesis [6]. Multiple Abeta oligomers, ranging from low-molecular weight oligomers to protofibrils and fibrils, apparently coexist in the brain tissue. However, the exact nature of these toxic aggregated Abeta peptides and their origin (intra- or extraneuronal or even plaque-derived) remains speculative. After the initial investigation by Podlisny et al. [15], a growing number of evidences strongly indicate that soluble Abeta forms, rather than insoluble species, including amyloid plaques, are the main toxic species associated with AD (see [1] and [5], for recent reviews). Subsequent studies by different independent groups, in an attempt to identify the precise soluble form that best correlated with cognitive impairment, have in fact reported a remarkable diversity of soluble aggregates in the interstitial fluid of human AD and transgenic model brains. Based on in vitro, using synthetic Abeta peptides, or ‘‘in vivo’’ experiments, different soluble Abeta oligomers, ranging from dimers to high-molecular weight aggregates (such as 12-mer, 24-mer, 32-mer, 150-mer) have been identified as the putative neurotoxic agents, disturbing the neurotransmission and causing neuronal death (see table 1 from [1]). The reasons for this apparent heterogeneity are not known but could derive from the different aggregation protocols of synthetic Abeta (for instance see [2] and [11]), the different transgenic models used or the different brain regions characterized. In this sense, it is particularly intriguing the quantitative and qualitative differences between soluble Abeta isolated by microdialysis and by homogenizationcentrifugation approaches, reported even by the same group [24]. Perhaps more relevant is the fact that, using brain samples from Alzheimer patients, a great disparity in the nature and quantities of soluble Abeta has been observed. For instance, relatively large amounts of monomeric and dimeric soluble Abeta (identified by western blots) have been reported by some groups [3,12,13,17,23,24] whereas others found small amounts (pg range, detected by dot blots or sensitive oligomeric specific ELISA assays) of soluble Abeta [4,21]. Among multiple causes, these differences between groups could reflect the absence of a unified isolation protocol for the extraction and characterization of soluble Abeta. In fact, many different homogenization (from manual to sonication) and centrifugation protocols have been used to isolate a theoretically similar extracellular soluble fraction. Considering that Abeta plaques constitute a large reservoir of different Abeta species, and that their number increased with the Soluble Abeta Peptides in AD Brain PLOS ONE | DOI:10.1371/journal.pone.0114041 December 8, 2014 2/17 pathology, strong homogenization protocols could indeed disturb the stability of plaques, and cause the release of different Abeta species. However, few (if any) control experiments were carried out to determine the extent of plaque disruption due to the isolation procedure. In the present study, we analysed the Abeta content in the soluble fractions of the hippocampus from human AD autopsy samples and transgenic models in order to investigate the possible repercussion of the homogenization protocols in the soluble fraction preparation. For that, we have directly compared the effect of homogenization using gentle conditions with sonication at different intensities. We concluded that strong homogenization, such as sonication, produced the release of Abeta peptides from Abeta plaques to soluble fractions. Materials and Methods Antibodies 82E1 and anti-soluble APPalpha antibodies were purchased from Immuno Biological Laboratories (IBL). 6E10 antibody was provided by Signet Laboratories. Anti-LC3B and anti-ATP synthase-beta were purchased from Cell Signaling Laboratories and BD Transduction Laboratories, respectively. Anti-mouse or antirabbit horseradish-peroxidase-conjugated secondary antibodies were purchased from Dako Denmark. Human samples The study was performed using 30 cases obtained from the BTIN-Tissue Bank for Neurological Research (Madrid, Spain) and from the Neurological Tissue Bank of IDIBELL-Hospital of Bellvitge (Barcelona, Spain), approved by the committee for human and animal use for research at Seville and Malaga Universities, Spain. The cases were scored for Braak stage for neurofibrillary tangles (II–VI). In Table 1 are detailed the age, Braak stage, sex and the delay post-mortem before the extraction of the samples. Preparation of human brain lysates Soluble (S1) fractions were prepared as described previously [9,10,19,22]. Briefly, frozen human hippocampal tissue was homogenized (1/5 w/v) using a manual Dounce homogenizer (10 strokes using pestle A (large clearance: Table 1. Human samples. mean Age ¡SD Sex Delay post-mortem mean h ¡SD BRAAK 84.0¡5.47 7 male - 4 female 6.2¡4.5 II 78.1¡11.99 4 male - 3 female 5.76¡5.44 III–IV 80.4¡10.21 4 male - 8 female 11.2¡4.0 V–VI doi:10.1371/journal.pone.0114041.t001 Soluble Abeta Peptides in AD Brain PLOS ONE | DOI:10.1371/journal.pone.0114041 December 8, 2014 3/17 0.114¡0.025 mm) and 10 strokes using pestle B (small clearance: 0.05¡0.025 mm), in Tris-buffered saline (TBS; 20 mM Tris-HCl, 140 mM NaCl, pH 7.5) containing protease and phosphatase inhibitors (Roche). Homogenates were then ultracentrifuged (4˚C during 60 min) at 100,0006g (TLA110 rotor, Optima MAX Preparative Ultracentrifuge, Beckman Coulter). The supernatant, which constitutes the S1 fractions, was collected, aliquoted and stored at 280˚C for further use. The remaining pellet (P1) was stored at 280˚C until needed. Sequential protein extraction For sequential extraction experiments, the P1 fraction was first resuspended (by Dounce homogenization; 10 strokes using pestle B) in RIPA buffer (1% CHAPS, 1% deoxycholate, 0.2% SDS, 140 mM NaCl, 10 mM Tris-HCl, pH 7.4, containing protease and phosphatase inhibitors), incubated for 30 min at 4˚C with agitation, and centrifuged at 30,0006g for 30 min at 4˚C. The supernatant constituted the S2 fraction. The pellet from this centrifugation was sequentially extracted (by incubating during 30 min, 4˚C followed by centrifugation 30,0006g for 30 min) using buffered-SDS 2% (2% SDS in 20 mM Tris-HCl, pH 7.4, 140 mM NaCl) (S3 fraction) and 4% SDS plus 8 M urea (P3 fraction). All isolated fractions were aliquoted and stored at 280˚C until use. Abeta quantification by sandwich ELISA The amount of soluble Abetax-40 or Abetax-42 peptides was determined in human soluble S1 fractions, using commercial sandwich ELISA (Human Abx-40 ELISA Kit, Invitrogen; Amyloid beta x-42 ELISA, DRG) following the manufacturer recommendations. For these experiments equivalent amount of proteins, from the different S1 fractions isolated from Braak II to Braak V–VI samples, were pooled. For each assay, 25 mg of protein from the pooled-soluble fractions was used. The ELISA experiments were repeated four times, in independent experiments, using duplicate or triplicate replicas. For oligomeric ELISA, we used 6E10-6E10 antibody pairs in conjunction with the Alpha-Lisa technology, developed by Perkin Elmer. This assay recognized synthetic Abeta dimer, used as standards, whereas synthetic monomeric Abeta42 produced no signal over the background. The lower limit sensitivity of this assay was 0.02 pg/ml. Preparation of PS1delta9xAPPswe cortical fractions All animal experiments were performed in accordance with the guidelines of the Committee of Animal Research of the University of Seville (Spain), University of Malaga (Spain) and the European Union Regulations. B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J (PS1xAPP) transgenic model was maintained in C57BL/6 genetic background for several generations (F10). Cortical samples from 12 months PS1xAPP transgenic mice were homogenized in TBS (1/ 10 w/v) containing protease and phosphatase inhibitor-cocktail (Roche), using Soluble Abeta Peptides in AD Brain PLOS ONE | DOI:10.1371/journal.pone.0114041 December 8, 2014 4/17 two different approaches: a) manual Dounce homogenizer (10 strokes with pestle A and 10 strokes with pestle B) or b) sonication (100 W ultrasonic processor UP100 H, Hielscher) for x4 or x8, 15 sec pulses at 4˚C. After homogenization, the different fractions were isolated as described in [17]. Briefly, homogenates were first centrifuged at 14,0006g (30 min, 4˚C). The supernatant (containing soluble and microsomal fraction) was then ultracentrifuged at 100,0006g (60 min, 4˚C) using a TLA110 rotor (Optima MAX Preparative Ultracentrifuge, Beckman Coulter). The supernatant of this ultracentrifugation constituted the TBS soluble fraction. The pellet, that constituted the microsomal fraction, was resuspended in TBS. On the other hand, pellets from the first centrifugation (membranes plus Abeta plaques) were sequentially solubilized using 2% buffered-SDS followed by SDS- Urea (4% SDS plus 8 M urea). PS1xAPP sequential protein extraction For these experiments, 12-month-old PS1xAPP cortical tissue was used. The TBS soluble fraction was isolated by ultracentrifugation as described above. For the sequential detergent-based extraction, the ultracentrifugation pellets were sequentially extracted using 2% CHAPS (in TBS), RIPA buffer, 2% SDS and finally 4% SDS-8 M urea. For each detergent treatment pellets were resuspended in the appropriate media, incubated for 30 min at 4˚C and centrifuged at 30,0006g (30 min, 4˚C). All fractions were stored at 280˚C for further use. Western blots Western blots were performed as described previously [9,10,19,22]. Briefly, for Abeta peptides, 10–20 mg (unless stated in text) of protein from the different samples were loaded on 16% SDS-Tris-Tricine-PAGE and transferred to PVDF (Immobilon-P Transfer Membrane, Millipore). For sAPPalpha, LC3 and ATPsynthase, 12% SDS-Tris-Glicine-PAGE was used and transferred to nitrocellulose (Optitran, GE Healthcare Life Sciences). Membranes were then blocked using 2% low-fat milk in TPBS (0.1% Tween-20, 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer , pH 7.4) and incubated overnight, at 4˚C, with the appropriate antibody. The membranes were then incubated with anti-mouse or anti-rabbit horseradish-peroxidase-conjugated secondary antibody (Dako) at a dilution of 1/10,000. The blots were developed using the Pierce ECL 2 Western Blotting Substrate detection method (0.5 pg, lower limit sensitivity; Thermo Scientific). The images were obtained with the luminescent image analyzer Image Quant LAS4000 mini (GE Healthcare Life Sciences) and then analyzed using PCBAS program. RNA extraction, retrotranscription and real-time RT-PCR Total RNA was extracted from cells (line N13) using Tripure Isolation Reagent (Roche). After isolation the RNA integrity was determined, by agarose gel Soluble Abeta Peptides in AD Brain PLOS ONE | DOI:10.1371/journal.pone.0114041 December 8, 2014 5/17 electrophoresis, resulting comparable in all samples. Total RNA was determined by spectrophotometric measures. Retrotranscription (RT) was performed using 4 mg of total RNA as template and High-Capacity cDNA Archive Kit (Ref.4368813, Applied Biosystems) following the manufacturer recommendations. For real time RT-PCR, 40 ng of cDNA were mixed with 2x Taqman Universal Master Mix (Ref.4369016, Applied Biosystems) and 20x Taqman Gene Expression assay probes (TNFalpha, Mm00443258_m1; IL1beta, Mm00434228_m1; IL6 Mm00446190_m1; GAPDH, Mm99999915_g1; 18s, Mm03928990; beta-actin, Mm00607939_s1; Applied Biosystems). PCR reactions were carried out in 96 well plates using either ABI Prism 7000 or 7900HT sequence detector systems (Applied Biosystems). The RTPCR conditions included two initial steps of 2 min at 50˚C to activate the polymerase followed by 10 min at 95˚C, and the subsequent 40 cycles of denaturation (95˚C, 15 sec) and annealing (60˚C, 1 min). The cDNA levels of the different samples were determined using GAPDH as housekeeper. Similar results were obtained using beta-actin or 18s rRNA (not shown). The amplification of the housekeeper was done in parallel with the gene to be analyzed, showing a similar amount of cDNA in all tested samples. All our data are expressed as the mean of at least three different measures [9], [10], [19], [22]. Amyloid plaque isolation Beta-amyloid plaques were isolated as described [18]. PS1xAPP (12 months of age) cortical samples were homogenized using Dounce as above and centrifuged at 2,0006g for 30 min (4˚C). The supernatant was further centrifuged for 1 hour at 20,0006g to collect the pellet, enriched in amyloid beta plaques. This pellet was resuspended in PBS, layered over a discontinuous sucrose gradient (0.4 ml of 1.0 M; 0.4 ml of 1.2, 0.8 ml of 1.4 and 0.4 ml of 2.0 M sucrose in PBS, pH 7.4) and centrifuged at 100,0006g (Optima MAX Preparative Ultracentrifuge, Beckman Coulter) for 1 h at 4˚C using a TLS-55 swing rotor. Then, fractions (from top) were removed and the 1.4–2.0 M sucrose interphase (enriched in amyloid plaques) was collected and diluted in PBS. The enriched amyloid fraction was centrifuged at 100,0006g for 1 h at 4˚C, and the pellet was collected and resuspended in PBS pH 7.4. The sample was separated in two aliquots, one was Dounce homogenated and the other one was subjected to sonication (8 pulses of 15 seconds). The two samples were centrifuged at 100,0006g for 1 h at 4˚C, and the supernatant and pellet were collected. N2a cultures and cell survival N2a cells were plated at 25,000 cells/cm2 and cultured in high glucose DMEM (Biowest)/OptiMEM (Gibco) (50–50%) supplemented with 2 mM glutamine (Biowest) and 5% (v/v) foetal bovine serum (Biowest) in the presence of penicillin (Biowest) and streptomycin (Biowest) (100 units/ml and 0.01 mg/ml, respectively) at 37˚C and 5% CO2. For survival experiments, the medium was changed Soluble Abeta Peptides in AD Brain PLOS ONE | DOI:10.1371/journal.pone.0114041 December 8, 2014 6/17 to high glucose DMEM and serum-deprived. The cells were then treated with different amounts of TBS fractions, from Dounce or sonication homogenization, during 24 hours. The cells survival was assayed by Flow Cytometry using the apoptosis detection kit ANNEXIN V-FITC (Immunostep). N13 culture and stimulation The N13 microglial cells were plated at 15,000 cells/cm2 and cultured in RPMI 1640 (Biowest) supplemented with 2 mM glutamine (Biowest) and 5% (v/v) foetal bovine serum (Biowest), in the presence of 100 U/ml penicillin and 100 mg/ ml streptomycin (Biowest) at 37˚C and 5% CO2. Cells were treated with different amounts of TBS soluble fractions, from Dounce or sonication, during 3 hours. Then, they were collected to isolate RNA. As controls, N13 cells were treated with equivalent volume of PBS. Statistical analysis Normality of data was first assessed by using Kolmogorov-Smirnov test. Normally distributed data were expressed and represented as mean ¡SD. For normally distributed data, means were compared using ANOVA followed by Tukey test (more than two groups) or t-test (for two group comparisons). Results Low content of soluble Abeta oligomeric in AD patients hippocampal formation We have first analyzed the Abeta content of soluble fractions prepared from the hippocampal formation (hippocampus proper, subiculum and entorhinal cortex), using Dounce homogenization and ultracentrifugation (see also [9,10,19,22]), of AD (Braak stage V–VI) and non-demented control (Braak stages II to IV) agematched autopsy samples. Quantitative analysis, using Abetax-42 or Abetax-40 sandwich ELISA (Fig. 1A), demonstrated the presence of very small amounts (pg/mg of protein) of soluble Abeta40-42 peptides in both AD and non-AD samples (see also [4] and [24]). In fact, Braak II samples displayed low levels of Abeta40 whereas Abeta42 was practically undetectable. As shown (Fig. 1A, a1 and a2), we observed a gradual and significant increase in both soluble species, Abeta42 and Abeta40, although more pronounced in the Abeta42 peptide, along the disease progression (3.1-fold in Braak III–IV and 12.5-fold in Braak V–VI). In consequence, the soluble Abeta42 peptide became equimolar with Abeta40 in demented patients (Fig. 1A, a3). Using the Abeta40 plus Abeta42 peptide to estimate the total soluble Abeta species, our soluble samples from AD brains contained approx 4 ng/g tissue of soluble Abeta40-42 peptides. This value was clearly lower to that reported by others [13]. Soluble Abeta Peptides in AD Brain PLOS ONE | DOI:10.1371/journal.pone.0114041 December 8, 2014 7/17 We next attempted to characterize the Abeta species using western blots. Importantly, neither monomeric/dimeric Abeta nor other oligomeric Abeta species could be detected in soluble (S1, Fig. 1B) or in vesiculated (S2, Fig. 1B) Braak VI fractions, as compared with Braak II samples. However, the monomericdimeric Abeta (and probably other oligomeric forms) were clearly visible when Abeta plaques were solubilized using 2% SDS (S3) and especially using 4% SDS/ 8M urea (P3) (Fig. 1B). Furthermore, even using a large amount of proteins (100 mg of soluble fraction, S1) in 6E10 immunoprecipitation assays (Fig. 1C), we were unable to detect any distinctive Abeta specie in Braak V–VI, compared with Braak II. Taken together, these data are consistent with the existence of low levels of soluble Abeta (pg levels), and the virtual absence of monomeric Abeta in the Figure 1. Low soluble Abeta levels were identified in hippocampal samples from age-matched controls (Braak II and III–IV) and demented patients (Braak V–VI). A: Soluble fractions from 11 Braak II, 7 Braak III–IV or 12 Braak V–VI samples were pooled and the Abeta x-42(a1) or x-40(a2) quantified by sandwich ELISA. Each assay was repeated 4 times in triplicate replicas (n54 per Braak stage). Although the level of Abeta species from all samples was low (pg/mg protein), there was a significant (showed in the figure) increased in Abetax-42 and Abetax-40 in Braak V–VI. In consequence, there was also an increase in Abeta42/40 ratio (a3) from Braak II to Braak V–VI. B: Western blots of soluble (S1) or detergent-based extracted proteins showed the absence, or below detection limits, of Abeta monomers or oligomers in soluble fractions. Hippocampal samples from Braak VI or Braak II individuals were sequentially extracted in parallel. No Abeta bands were detected in S1 or CHAPS fractions. Monomeric, dimeric and other oligomeric species were clearly identifiedin 2% SDS and 4% SDS/8M urea samples. C: (left panel) One hundred micrograms of proteins from pooled soluble (S1) Braak II or Braak V–VI fractions were subjected to immunoprecipitation using 6E10 antibody. The immunopellets were analyzed by western blots using 6E10 (not shown) or 82E1. No specific bands, by comparison between Braak II and V–VI, were observed. Control experiments (right panel) demonstrated that 0.5 pg of synthetic Abeta42 could clearly be observed by our western blot system. doi:10.1371/journal.pone.0114041.g001 Soluble Abeta Peptides in AD Brain PLOS ONE | DOI:10.1371/journal.pone.0114041 December 8, 2014 8/17 soluble fraction of AD samples. We cannot discard the presence of minute amounts of monomeric Abeta (below the detection limits of our western blots, 0.5 pg, see Fig. 1C) or small quantities of oligomeric Abeta, not detected by our assays. In any case, these data are in clear contrast with those reported recently [13,14,16,17,23]. The homogenization procedure largely determined the content of Abeta species in the soluble fraction While multiple reasons could explain the different levels of soluble Abeta reported in human AD samples, the initial homogenization of the tissue could be crucial to maintain the integrity of the Abeta plaques. A strong (aggressive) treatment of the tissue could disrupt the aggregated Abeta from plaques and, in consequence, contaminate all fractions with artifactual, plaque-derived, soluble Abeta species. To evaluate this possibility, we have directly compared the effect of homogenization using manual Dounce (see Methods) with sonication at different intensities. As a natural rich source of human Abeta, we have used 12 month-old cerebral cortex from PS1xAPP double transgenic mice. At this age, this model displayed the presence of abundant Abeta plaques in the cerebral cortex (not shown). After homogenization, the TBS soluble fraction (equivalent to S1, see above) and the microsomal (equivalent to the dispersible fraction reported by [16,17]) fractions were isolated by ultracentrifugation (see Methods). The Abeta from plaques were then extracted by sequential solubilization using 2% SDS and 4% SDS plus 8 M urea (following [16,17] protocol). As expected from our previous work [10,19,22] (see Fig. 2A), the monomeric and oligomeric Abeta were preferentially observed in membrane/plaque associated fractions, extracted with 2% SDS and 4% SDS/8M urea. These fractions released a relatively large amount of monomers, dimers and other oligomeric Abeta species. However, as also reported previously [10,19,22], in 6–12 months old PS1xAPP mice, these species were absent (or below the detection limit of our western blots) in the TBS soluble fraction or in the microsomal fraction (see Fig. 2A). The lack of oligomeric Abeta in the TBS fraction was also probed by using a 6E10-6E10 oligomeric ELISA (AlphaLisa, Perkin Elmer) assay (Fig. 2B). On the contrary, when the same cortical tissue was subjected to sonication, a pulse-dependent redistribution of Abeta in all soluble fractions was observed. As shown, the TBS soluble fraction was contaminated with relatively high levels of monomeric Abeta (Fig. 2A). Furthermore, we also observed the presence of different putative Abeta oligomers, such as dimers, trimers, and low molecular weight-Abeta in these contaminated soluble fractions (the presence of such oligomers was really high after 8 pulses of sonication, see Fig. 2A). These Abeta oligomers were also clearly detected by 6E10-6E10 oligomeric Abeta ELISA (Fig. 2B). Similarly, the microsomal fractions were also highly contaminated with different Abeta species. Concomitantly, we also observed a decrease in the Abeta content from plaque rich fractions (2% SDS and 4% SDS/8M urea extracted pellets). Therefore, sonication (even using low Soluble Abeta Peptides in AD Brain PLOS ONE | DOI:10.1371/journal.pone.0114041 December 8, 2014 9/17 Author Contributions Conceived and designed the experiments: JV AG MV JCD. Performed the experiments: SJ VN MT MSM JM. Analyzed the data: JV AG MV JCD. Contributed reagents/materials/analysis tools: JV AG. 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