Article3 September 2010free access Neurotoxicity of Alzheimer's disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio Inna Kuperstein Inna Kuperstein Department for Molecular and Developmental Genetics, Flanders Institute for Biotechnology (VIB), Leuven, Belgium Center for Human Genetics, KULeuven, Leuven, BelgiumPresent address: Institut Curie, Département de Transfert, and INSERM, U900, Paris F-75248 France Search for more papers by this author Kerensa Broersen Kerensa Broersen Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Iryna Benilova Iryna Benilova Department for Molecular and Developmental Genetics, Flanders Institute for Biotechnology (VIB), Leuven, Belgium Center for Human Genetics, KULeuven, Leuven, Belgium IMEC, Bioelectronic Systems Group, Heverlee, Belgium Search for more papers by this author Jef Rozenski Jef Rozenski Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Leuven, Belgium Search for more papers by this author Wim Jonckheere Wim Jonckheere Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Maja Debulpaep Maja Debulpaep Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Annelies Vandersteen Annelies Vandersteen Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Ine Segers-Nolten Ine Segers-Nolten Faculty of Science and Technology, Nanobiophysics, MESA+ Institute for Nanotechnology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands Search for more papers by this author Kees Van Der Werf Kees Van Der Werf Faculty of Science and Technology, Nanobiophysics, MESA+ Institute for Nanotechnology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands Search for more papers by this author Vinod Subramaniam Vinod Subramaniam Faculty of Science and Technology, Nanobiophysics, MESA+ Institute for Nanotechnology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands Search for more papers by this author Dries Braeken Dries Braeken IMEC, Bioelectronic Systems Group, Heverlee, Belgium Search for more papers by this author Geert Callewaert Geert Callewaert IMEC, Bioelectronic Systems Group, Heverlee, Belgium Search for more papers by this author Carmen Bartic Carmen Bartic IMEC, Bioelectronic Systems Group, Heverlee, Belgium Department of Physics and Astronomy, Laboratory of Solid State Physics and Magnetism, KULeuven, Belgium Search for more papers by this author Rudi D'Hooge Rudi D'Hooge Laboratory of Biological Psychology, KULeuven, Leuven, Belgium Search for more papers by this author Ivo Cristiano Martins Ivo Cristiano Martins Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, BelgiumPresent address: Biomembranes Unit, Institute de Medicina Molecular (IMM), Av. Prof. Egas Moniz, Lisboa, Portugal Search for more papers by this author Frederic Rousseau Corresponding Author Frederic Rousseau Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Joost Schymkowitz Corresponding Author Joost Schymkowitz Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Bart De Strooper Corresponding Author Bart De Strooper Department for Molecular and Developmental Genetics, Flanders Institute for Biotechnology (VIB), Leuven, Belgium Center for Human Genetics, KULeuven, Leuven, Belgium Search for more papers by this author Inna Kuperstein Inna Kuperstein Department for Molecular and Developmental Genetics, Flanders Institute for Biotechnology (VIB), Leuven, Belgium Center for Human Genetics, KULeuven, Leuven, BelgiumPresent address: Institut Curie, Département de Transfert, and INSERM, U900, Paris F-75248 France Search for more papers by this author Kerensa Broersen Kerensa Broersen Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Iryna Benilova Iryna Benilova Department for Molecular and Developmental Genetics, Flanders Institute for Biotechnology (VIB), Leuven, Belgium Center for Human Genetics, KULeuven, Leuven, Belgium IMEC, Bioelectronic Systems Group, Heverlee, Belgium Search for more papers by this author Jef Rozenski Jef Rozenski Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Leuven, Belgium Search for more papers by this author Wim Jonckheere Wim Jonckheere Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Maja Debulpaep Maja Debulpaep Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Annelies Vandersteen Annelies Vandersteen Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Ine Segers-Nolten Ine Segers-Nolten Faculty of Science and Technology, Nanobiophysics, MESA+ Institute for Nanotechnology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands Search for more papers by this author Kees Van Der Werf Kees Van Der Werf Faculty of Science and Technology, Nanobiophysics, MESA+ Institute for Nanotechnology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands Search for more papers by this author Vinod Subramaniam Vinod Subramaniam Faculty of Science and Technology, Nanobiophysics, MESA+ Institute for Nanotechnology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands Search for more papers by this author Dries Braeken Dries Braeken IMEC, Bioelectronic Systems Group, Heverlee, Belgium Search for more papers by this author Geert Callewaert Geert Callewaert IMEC, Bioelectronic Systems Group, Heverlee, Belgium Search for more papers by this author Carmen Bartic Carmen Bartic IMEC, Bioelectronic Systems Group, Heverlee, Belgium Department of Physics and Astronomy, Laboratory of Solid State Physics and Magnetism, KULeuven, Belgium Search for more papers by this author Rudi D'Hooge Rudi D'Hooge Laboratory of Biological Psychology, KULeuven, Leuven, Belgium Search for more papers by this author Ivo Cristiano Martins Ivo Cristiano Martins Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, BelgiumPresent address: Biomembranes Unit, Institute de Medicina Molecular (IMM), Av. Prof. Egas Moniz, Lisboa, Portugal Search for more papers by this author Frederic Rousseau Corresponding Author Frederic Rousseau Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Joost Schymkowitz Corresponding Author Joost Schymkowitz Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium Vrije Universiteit Brussel (VUB), Brussels, Belgium Search for more papers by this author Bart De Strooper Corresponding Author Bart De Strooper Department for Molecular and Developmental Genetics, Flanders Institute for Biotechnology (VIB), Leuven, Belgium Center for Human Genetics, KULeuven, Leuven, Belgium Search for more papers by this author Author Information Inna Kuperstein1,2,‡, Kerensa Broersen3,4,‡, Iryna Benilova1,2,5,‡, Jef Rozenski6, Wim Jonckheere3,4, Maja Debulpaep3,4, Annelies Vandersteen3,4, Ine Segers-Nolten7, Kees Van Der Werf7, Vinod Subramaniam7, Dries Braeken5, Geert Callewaert5, Carmen Bartic5,8, Rudi D'Hooge9, Ivo Cristiano Martins3,4, Frederic Rousseau 3,4, Joost Schymkowitz 3,4 and Bart De Strooper 1,2 1Department for Molecular and Developmental Genetics, Flanders Institute for Biotechnology (VIB), Leuven, Belgium 2Center for Human Genetics, KULeuven, Leuven, Belgium 3Switch Laboratory, Flanders Institute for Biotechnology (VIB), Brussels, Belgium 4Vrije Universiteit Brussel (VUB), Brussels, Belgium 5IMEC, Bioelectronic Systems Group, Heverlee, Belgium 6Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Leuven, Belgium 7Faculty of Science and Technology, Nanobiophysics, MESA+ Institute for Nanotechnology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands 8Department of Physics and Astronomy, Laboratory of Solid State Physics and Magnetism, KULeuven, Belgium 9Laboratory of Biological Psychology, KULeuven, Leuven, Belgium ‡The authors contributed equally to this work *Corresponding authors: Switch Laboratory, Flanders Institute for Biotechnology (VIB) and Vrije Universiteit Brussel (VUB), Pleinlaan 2, Brussels 1050, Belgium. Tel.: +322 629 1025; Fax: +322 629 1942; E-mail: [email protected] Laboratory, Flanders Institute for Biotechnology (VIB) and Vrije Universiteit Brussel (VUB), Pleinlaan 2, Brussels 1050, Belgium. Tel.: +322 629 1025; Fax: +322 629 1942; E-mail: [email protected] for Human Genetics, Flanders Institute for Biotechnology (VIB) and KULeuven, Herestraat 49, Leuven 3000, Belgium. Tel.: +321 634 6227; Fax: +321 634 7181; E-mail: [email protected] The EMBO Journal (2010)29:3408-3420https://doi.org/10.1038/emboj.2010.211 Present address: Institut Curie, Département de Transfert, and INSERM, U900, Paris F-75248 France PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The amyloid peptides Aβ40 and Aβ42 of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aβ42 seems more pathogenic than Aβ40, the reason for this is not well understood. We show here that small alterations in the Aβ42:Aβ40 ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ42:Aβ40 ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti-amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non-toxic intermediates. Introduction Amyloid β (Aβ) peptides generated from the amyloid precursor protein (APP) by β- and γ-secretase-mediated cleavage (Annaert and De Strooper, 2002) are thought to have an important function in the neurodegenerative process in Alzheimer's disease (AD) (Hardy and Selkoe, 2002). γ-Secretase cleavage of APP generates a heterogeneous mixture of Aβ peptides varying in length at their carboxytermini (Sato et al, 2003; Qi-Takahara et al, 2005; Kakuda et al, 2006). Additional heterogeneity is generated at the aminoterminus by aminopeptidases, glutaminylcyclases and other modifications (Pike et al, 1995; Saido et al, 1996) (reviewed in De Strooper, 2010). It has been proposed that some of these variations might contribute to the neurotoxic properties of Aβ peptides (Schilling et al, 2008). The major Aβ species recovered from serum, cerebrospinal fluid and cell culture supernatants is 40 amino acids long (Aβ40) (Haass and Selkoe, 1993; Scheuner et al, 1996). Interest in a second peptide, Aβ42, which is detected at about 10-fold lower levels, was strongly stimulated by the observation that familial AD causing mutations in the APP gene and/or in the gene encoding the γ-secretase complex component presenilin increased the relative production of Aβ42 relative to Aβ40 (Suzuki et al, 1994; Duff et al, 1996; Scheuner et al, 1996). We reported earlier (Bentahir et al, 2006) that clinical mutations in presenilin do not necessarily increase the production of Aβ, but that they mainly affect the spectrum of the Aβ peptides generated by γ-secretase. As patients with presenilin mutations present an early and aggressive form of the disease, it seems then logical to propose that the absolute quantity of Aβ peptides produced in the brain might be less important than the quality of the Aβ peptides (reflected in a changed Aβ42 to Aβ40 ratio) for the generation of elusive toxic Aβ species (De Strooper, 2007). The implications of such hypothesis for current efforts in drug development is important because lowering the absolute amounts of Aβ in patients would then be less crucial than the restoration of the correct ratios of Aβ peptides. Earlier studies have already provided evidence that Aβ40 and Aβ42 affect each other's aggregation rates and toxic effects (Snyder et al, 1994; Frost et al, 2003; Yoshiike et al, 2003; Wang et al, 2006; Kim et al, 2007; Yan and Wang, 2007; Jan et al, 2008). Generally, it is found that Aβ42 has fast aggregation kinetics, which can be inhibited by Aβ40 in a concentration-dependent manner. Interesting in vivo studies have further shown that increased levels of Aβ40 peptides in the brain actually might have a protective effect (Wang et al, 2006; Kim et al, 2007). In the past, a lot of attention has gone to the accumulation of Aβ in plaques and the relationship between amyloid plaques and AD. However, little or no correlation was found between the total burden of Aβ peptide deposited into plaques in the brain and the degree of neurodegeneration in the patients (Terry et al, 1991; Price and Morris, 1999). More recently, this discrepancy has been confirmed with modern amyloid imaging techniques (Aizenstein et al, 2008; Reiman et al, 2009). Obviously, it is possible that these patients are in a preclinical phase of the disease, and follow-up studies are underway to investigate this. Nevertheless, these observations support the concept that the amyloid fibrils are biologically largely inert and that not all conformations of Aβ are equally toxic (Martins et al, 2008; Shankar et al, 2008). A series of intermediate soluble aggregates of Aβ peptides, such as ‘Aβ-derived diffusible ligands’ (ADDLs) (Lambert et al, 1998) or ‘natural toxic oligomers’ (Walsh et al, 2002), have been identified. The mechanism of their neurotoxic activity remains not only subject of intense investigation, but also the precise conformation(s) of the toxic species remains uncertain (Kayed et al, 2003; Hepler et al, 2006). Dimers were proposed to potently disrupt synaptic plasticity (Klyubin et al, 2008; Shankar et al, 2008), an Aβ species of 56 kDa has been found neurotoxic in Tg2576 mice (Lesne et al, 2006), lipid-induced oligomers from mature fibrils (Martins et al, 2008), ADDLs (Lambert et al, 1998; Gong et al, 2003; Lacor et al, 2007) and annular assemblies (Lashuel et al, 2002) were shown to exert neurotoxic effects, affect synapse function and even memory formation in mice. It should be noted that in many of the publications, the identified toxic species are presented as stable, defined structures, although it seems logical to assume that their assembly and disassembly is a dynamic and continuous process, at least in the initial stages, and the alternative possibility that toxicity is present over a series of conformers or sizes should not be disregarded (Hepler et al, 2006; Martins et al, 2008; Ono et al, 2009). Toxicity seems to be higher with tetramers than dimers for instance (Ono et al, 2009). The question is thus how biophysical parameters influence this process in vivo and affect the relative distribution of Aβ species over toxic and non-toxic conformations over time. Given the complexity of the biophysical environment in which Aβ aggregation occurs in vivo, such question is extremely difficult to address. Nevertheless, it is possible to analyse the dynamic features of this process in simplified and controlled conditions in vitro, and to evaluate the effect of the relative concentrations of Aβ40 and Aβ42 to the generation of neurotoxic species over time. We hypothesized here that the early onset of AD by APP and/or presenilin mutations that increase the Aβ42:Aβ40 ratios can be explained by interactions between Aβ40 and Aβ42, which provide stability to intermediate, neurotoxic species. We used biophysical methods and a novel cellular assay to analyse the establishment of neurotoxicity over time in different Aβ mixtures. We found that very minor changes in the relative amount of Aβ42 versus Aβ40 (Aβ42:Aβ40) has dramatic effects on the dynamic behaviour of toxic Aβ species. Our findings provide an important biological addition to the original ‘Aβ42 seeding hypothesis’ (Jarrett and Lansbury, 1993), which focused on amyloid fibril formation. These dynamic oligomeric species exhibit initial synaptotoxicity and cause later neurotoxicity in primary hippocampal neurons and affect memory formation in mice, underlining their potential importance for the understanding of AD. Results As the objective of this study was to investigate how Aβ40 and Aβ42 affect each others’ biophysical and biological properties, it was important to prepare a pre-aggregate-free Aβ solution and to validate the mixtures using mass spectrometry (Supplementary Figure) and anti-Aβ40- and anti-Aβ42-specific antibodies (Supplementary Figure 1E). We found that the sequential treatment of 1,1,1,3,3,3-hexafluor-2-propanol (HFIP)-Aβ films (rPeptide) with HFIP, dimethyl sulphoxide (DMSO) and then removal of DMSO using a desalting column provide excellent results with mixtures of primarily monomeric peptides in the appropriate relative amounts (Supplementary Figure 1, see also Materials and methods). Fourier transform infrared (FTIR) spectroscopy validated the complete removal of all HFIP and DMSO (not shown). Aggregation rate of Aβ peptides is strongly influenced by the ratio Aβ42:Aβ40 Aβ peptide was incubated at a concentration of 50 μM in 50 mM Tris–HCl, 1 mM EDTA, pH 7.5 at 25°C. The aggregation process of a range of Aβ42:Aβ40 ratios (10:0 to 0:10) tested by Thioflavin T (ThT) fluorescence yielded a typical sigmoidal curve as generally observed for aggregating proteins and peptides (Figure 1A shows four examples) (Harper and Lansbury, 1997). The formation of an Aβ nucleus, which is not reactive with the fluorescent ThT probe during the so-called ‘lag phase’, is followed by rapid elongation of ThT-positive Aβ aggregates to form fibrils (the ‘elongation phase’). Both the length of the lag phase and the rate of aggregation were affected by the ratio of Aβ42:Aβ40 (Figure 1B and C). The lag phase for pure Aβ40 alone was ∼2.5 h (±0.3 h). Addition of 10% Aβ42 (Aβ42:Aβ40=1:9) resulted in a small, but reproducible increase in the lag phase (∼2.9±0.3 h) (Figure 1B). A further increase in the Aβ42:Aβ40 ratio decreased paradoxically the length of the lag phase to ∼0.5±0.01 h. From a ratio of 3:7 onwards, no difference was observed compared with Aβ42 alone. The elongation rate was fastest for pure Aβ40 and was slowed down by addition of Aβ42 (Figure 1C). Remarkably, judging from the lag phase of aggregation, the 1:9 and the 3:7 ratio showed two opposite ends of the spectrum (Figure 1B). These ratios, in addition to 10:0 and 0:10 were selected for our further studies. The choice for 3:7 can be also rationalized as it reflects roughly the ratio of Aβ42 and Aβ40 in patients with familial AD (Duff et al, 1996; Mann et al, 1996; Scheuner et al, 1996; Citron et al, 1997). Thus, both time and the Aβ42:Aβ40 ratios are two important parameters when considering the biophysical properties of Aβ, and we decided to investigate how these parameters determine Aβ-oligomer toxicity. We describe in the rest of the paper the different Aβ mixtures as an Aβ42:Aβ40 ratio, incubated for an indicated time at an Aβ concentration of 100 μM in 50 mM Tris–HCl, 1 mM EDTA, pH 7.5 at 25°C. Thus, (3:7, 2 h) means a ratio of three Aβ42 versus seven Aβ40 peptides incubated for 2 h under given buffer conditions before addition to the cell culture. Final concentration of Aβ in cell culture is in most experiments 1 μM apart from a few in which 10 μM was used as indicated. On the basis of our further experiments, it appeared that at any time point only a small fraction of the peptides is in a toxic active conformation. Figure 1.Aβ42 determines the kinetics of Aβ aggregation. (A) Aggregation kinetics of 50 μM Aβ ratios in 50 mM Tris, 1 mM EDTA at 25°C for 6 h by Thioflavin T (ThT) assay shows how the ratio of Aβ42:Aβ40 (e.g. 3:7) influences the aggregation kinetics. The colour codes are maintained in all following figures. (B) Quantitative analysis of lag phase, showing the time (hours) of the initial part of curves as in (A), during which no increase in ThT fluorescence signal is detected using different Aβ42:Aβ40 ratios as indicated. (C) Quantitative analysis of the elongation rate, derived from (A) as rate of fluorescence change, which is the slope of the linear phase of exponential growth in (A), using different Aβ ratios as indicated. Numbers are averages of three independent experiments. Error flags indicate s.d. calculated over the three independent experiments. Statistical significance of the results was established by P-values using paired two-tailed t-tests, and only shown for the four ratios further studied in the text. Statistical significance levels were in (B, C): *P<0.005, **P<0.001, ***P<0.0001. P-value of Aβ40 (0:10) and (1:9)=0.09, and P-value of Aβ40 (0:10) and (1:9)=0.0058 in panels B and C, respectively. Download figure Download PowerPoint The Aβ42:Aβ40 ratio is a driver of acute synaptic alterations One problem with Aβ toxicity assays is the delay between the actual preparation of the oligomer samples used for the biophysical analysis and the moment when the biological read out becomes available to assess the neurotoxicity. We, therefore, sought to set up an assay that allows verifying biological effects of Aβ preparations within the time frame of the biophysical experiments. We plated mouse hippocampal neurons on microelectrode array (MEA)-based chips (Figure 2A) (Stett et al, 2003) and recorded spontaneous firing rates in the neuronal networks before and after treatment with Aβ mixtures at a final concentration of 1 μM. Representative traces of responses of cultures treated with different Aβ mixtures are displayed in Figure 2B. Interestingly, treatment with pure Aβ40 mixtures (0:10, 2 h) appeared to enhance synaptic activity measured as spontaneous firing rate, whereas (1:9, 2 h) mixtures had no effect on spontaneous synaptic activity (Figure 2B and C). In contrast, Aβ42 alone (10:0, 2 h) or with Aβ40 (3:7, 2 h) readily suppressed spontaneous neuronal activity within 40 min after addition of the peptides (Figure 2B and C). As a control of the oligomers species status during the course of the recording, we followed changes in fluorescent ThT emission over 40 min of Aβ mixtures at 1 μM in neuronal culture medium at 37°C, which mimics the conditions of the cell culture experiments (Figure 2D). The Aβ mixtures appeared stable over the time frame of the experiment at least as far as it concerns ThT incorporation. We next assayed how different Aβ42:Aβ40 ratios evolve over time with regard to synaptotoxic properties. Aβ42:Aβ40 ratio mixtures were incubated for 0, 1.5, 4, 6 and 20 h before addition to the cultures. Synaptic firing rates were recorded for 40 min as above using MEA. Figure 2E shows that synaptic effects were little after treatment of the cells with mixtures of Aβ shortly after dissolving them in buffer (t=0 h, Figure 2E). Toxicity was, however, already significantly high after 1.5 h of aggregation in the (3:7, 1.5 h) and the (10:0, 1.5 h) mixtures. The synaptotoxic potential in the (3:7) and (10:0) ratios remained stable up to 20 h (Figure 2E). Remarkably, 1:9 or 0:10 ratios did not result in major synaptic effects at any incubation point (Figure 2E). To validate the findings, we performed double immunostaining for the synaptic marker synaptophysin and Aβ oligomers using the A11 antibody (Kayed et al, 2003). Figure 2F shows that Aβ (3:7) and Aβ (10:0) mainly co-localized with the synaptic marker, whereas staining was not observed with the (1:9) and (0:10) ratio. Extensive washing of the neurons to remove Aβ species did not interfere with consecutive A11 staining, indicating the rather irreversible nature of the binding of these synaptic active species to the neurons (not shown). Figure 2.Mixed Aβ oligomers result in a rapid synaptotoxic response in primary neurons. (A) Neurons stained with Fluo-4 were cultured 8 days in vitro (DIV) on the MEA chip. (B) Firing pattern of neurons from representative electrodes at 0, 10, 20 and 40 min treatment with different Aβ ratios prepared as indicated (e.g. 0:10, 2 h means an Aβ42:Aβ40 ratio of 0 versus 10, and 2 h means 2 h incubation before addition to the culture). Note the significantly decreased firing rate and amplitude for Aβ42:Aβ40 ratios 10:0 and 3:7 after 20 min of treatment. (C) Spontaneous electrical synaptic activity recordings of hippocampal neurons during 40 min of treatment with 1 μM Aβ ratios incubated for 2 h prior to the addition to cells. Values are per cent of initial firing rate±s.e.m. of 3–5 independent experiments. Statistical significance (unpaired two-tailed t-test) of the data versus control is indicated by *P<0.01 or **P<0.001 in the figure. Notice that a strong reduction in spontaneous synaptic firing versus correspondent control (buffer-treated chips) can be observed after 20 and 40 min in the medium of the 3:7 and 10:0 Aβ42:Aβ40 ratios. (D) Oligomers dissolved in culture medium remain stable with regard to ThT tinctorial properties for at least 40 min. Aβ ratios were prepared and at specific time intervals of incubation, that is 0, 1.5, 4 and 22 h, Aβ aliquots were removed and diluted to 1 μM in cell culture medium containing ThT. The stability of the aggregates at 37°C in cell culture medium was deduced from the stability of the ThT signal over 40 min. Blue bars represent different Aβ42:Aβ40 ratios as indicated in the figure. Compare signals directly upon dilution into cell culture medium (‘0 min’) at 37°C with signals obtained after 40 min incubation at 37°C (‘40 min’). Values are averages of three experiments. (E) Different ratios of Aβ peptides were generated and added to neuronal cultures diluted to a final concentration of 1 μM, either immediately (0 h) or after 1.5 h; 4 h, 6 h or 20 h of incubation. Synaptotoxicity was measured by recording a decreased rate of firing 40 min after adding the Aβ mixtures to the neurons. Statistical significance levels determined as a function of s.e.m.: ***P<0.0001, n=6 chips, **P<0.001, n=3 chips, *P<0.01, n=3 chips, difference between 6 h ratio (3:7) and (10:0): *P<0.012 (unpaired two-tailed t-test). (F) Synaptic localization of mixed Aβ oligomers. Fluorescence microscopy images of hippocampal neurons stained for synaptophysin (red) and Aβ oligomers (A11 antibody) (green) after 1 h treatment with 1 μM of the indicated Aβ ratios, incubated for 2 h prior the addition to cells. Right panel: magnification of selected region stained with synaptophysin (red) and Aβ oligomers (A11 antibody, green). Oligomeric Aβ co-localizes with synapses. (G) Rescue of spontaneous electrical synaptic activity after the treatment with ratio (10:0, 2 h) in the presence or absence of anti-oligomer A11 or anti-Aβ 6E10 antibody, final concentration 10 μg ml–1. Values are per cent of initial firing rate±s.e.m., of three independent experiments, except for the control with non-specific antibody, which was performed only once. (H) Example of firing recovery after treatment with 10 μM (10:0, 2 h). Raw data streams are shown in black, and corresponding spike shapes are in red. The treatment completely inhibited spontaneous activity in 6 min. Then the medium was refreshed, and signals were measured after overnight recovery (18 h). Note partial restoration of initial firing profile along with appearance of another spike population endowed with slightly different waveform and amplitude. Spike sorting is performed in MC Rack software. Download figure Download PowerPoint To prove Aβ specificity of the observed effects, we pre-incubated cells with anti-oligomer A11 or anti-Aβ antibody 6E10 before treatment with Aβ (10:0, 2 h) ratio. The alterations in the synaptic activity by toxic intermediates are indeed Aβ specific, as cells pre-incubated with antibodies are no longer susceptible to the neurotoxic effects (Figure 2G). We also further explored the reversibility of these Aβ effects on synapti