The Anti-Prion Antibody 15B3 Detects Toxic Amyloid-β Oligomers

Materials

15B3 was obtained from Prionics (now Thermo Fisher Scientific, Rockford, IL USA). Three batches of 15B3 were used: most of the studies were carried out with batch # 071114A, with a nominal concentration of 5 mg/mL; when indicated, batches # 110531 (0.9 mg/mL) and # 061013 (0.8 mg/mL) were also used.

Control mouse IgM was from Thermo Fisher Scientific, Rockford, IL, USA; anti-Aβ monoclonal antibody 4G8 was from Covance, Princeton, NJ, USA; anti-Aβ oligomer polyclonal antibodies OC and A11 were from Merck Millipore, Darmstadt, Germany; mouse monoclonal anti-Aβ 6E10 was from Covance, Emerville, CA, USA; rabbit anti-β-tubulin was from Sigma, St. Louis, MO, USA and guinea pig anti-Bassoon was from Synaptic System, Gottingen, Germany.

Aβ₄₂ preparation

Depsi-Aβ₄₂ was synthesized in-house, as previously described [14]. In comparison with the highly aggregating Aβ₄₂, the more soluble depsi form has a much lower propensity for spontaneous aggregation [14, 15] preventing the formation of seeds in solution. Aβ₄₂ was then obtained from the depsi-peptide by a “switching” procedure involving a change in pH [16]. The solution was diluted in 10 mM PBS, pH 7.4, to a final Aβ₄₂ concentration of 100 μM. This procedure allows the preparation of reproducible, seed-free solutions of monomeric Aβ₄₂, as shown by circular dichroism, size exclusion chromatography, and SPR [7, 14]. To obtain aggregated Aβ₄₂, the solution was incubated at 25°C in quiescent conditions for different times. We used freshly prepared solutions (t = 0) to have Aβ₄₂ monomers only, or Aβ₄₂ solutions incubated for 5 h (t = 5 h) for maximal oligomer enrichment [7].

SPR studies

The SPR apparatus we employed (ProteOn XPR36 Protein Interaction Array System; Bio-Rad) has six parallel flow-channels that can be used to uniformly immobilize six different ligands on the sensor surface. The fluidic system can automatically rotate 90° so up to six different analytes can be injected, allowing simultaneous monitoring of up to 36 individual molecular interactions in a single run on a single chip [17]. 15B3 was immobilized in a flow channel of GLC sensor chips (Bio-Rad) using amine coupling chemistry, as described [7]. Briefly, after surface activation, the antibody (30 μg/mL in 10 mM acetate buffer, pH 5.0) was injected for 5 min at a flow rate of 30 μL/min, and the remaining activated groups were blocked with ethanolamine, pH 8.0. The final immobilization level was about 7000 resonance units (1 RU = 1 pg of protein/mm²). A “reference” surface was prepared in parallel using the same immobilization procedure without adding the antibody. Recombinant mouse PrP, prepared as previously described [18, 19], was also immobilized by amine coupling chemistry, as previously described [20] (immobilization  3000 RU). After rotation of the microfluidic system, Aβ₄₂ was injected for 2 min at a flow rate of 30 μL/min. Dissociation was measured in the next 11 min. The running buffer, also used to dilute the samples, was 10 mM PBS containing 0.005% Tween 20 (PBST). Assays were run at 25°C. The sensorgrams (time course of the SPR signal in RU) were normalized to a baseline of 0. The signal in the surfaces immobilizing the antibody was corrected by subtracting the nonspecific response observed in the reference surface.

Enzyme-linked immunosorbent assay (ELISA)

Synthetic Aβ₄₂ was dissolved in PBS to a final concentration of 100 μM and incubated at 25°C in quiescent conditions. Aliquots were taken at different incubation times (from 0 to 72 h), diluted to 1 μM in PBS, loaded in triplicate on a 15B3-precoated 96-well microplate (Prionics, now Thermo Fisher Scientific, Rockford, IL, USA) and incubated 1 h at room temperature under shaking. The plate was washed four times with PBS containing 0.005% Tween-20 (PBST) and Aβ₄₂ oligomers were detected with HRP-labeled 4G8 (Covance), 1 : 50 000 in PBST, incubated for 1 h at room temperature, protected from light. After four washes with PBST, Luminata Forte Western HRP Substrate (Millipore) was added and luminescence was measured with a F500 Infinite plate reader (Tecan Italia Srl, Italy).

Kinetics of fibril formation

The kinetics of Aβ₄₂ aggregation was monitored using an in situ Thioflavin-T (ThT) fluorescence assay based on the increase of the fluorescence signal of ThT when bound to β-sheet-rich structures. Aβ₄₂, 4 μM, was incubated, with and without 15B3 or control IgM, under quiescent conditions at 37°C in microplate wells (Microplate Corning 3881, 96-well, low-binding, Corning Incorporated Life Sciences, Acton, MA) in the presence of 20 μM ThT (100 μL solution per well). ThT fluorescence was measured every 2.5 min using an F500 Infinity plate reader (Tecan Italia Srl, Italy). The dye was excited at 448 nm (bandwidth 7 nm) and the emission measured at 485 nm (bandwidth 20 nm).

Aβ₄₂ binding to neurons

Primary hippocampal neurons were established from Sprague Dawley E18 fetal rats (Charles River Italia). The experimental procedures followed the guidelines established by European (Directive 2010/63/EU) and Italian legislation (L.D. no. 26/2014). They were reviewed and approved by the Animal Welfare Committee of the University of Milan and by the Italian Ministry of Health. Briefly, dissociated cells were plated onto poly-L-lysine-treated coverslips at 520 cells/mm² density and maintained in Neurobasal medium with 2% B27 supplement and 2 mM glutamine (neuronal medium). 12–15 DIV hippocampal neurons were exposed to 1 μM Aβ₄₂ monomers or Aβ₄₂ oligomers for 1 h in neuronal medium. In one set of experiments, Aβ₄₂ oligomers were preincubated with 10 nM 15B3 or control IgM for 30 min before being administered to neurons. Neurons were then washed, fixed with 4% paraformaldehyde, and immunostained using the following antibodies: mouse monoclonal anti-Aβ 6E10, rabbit anti-β-tubulin and guinea pig anti-Bassoon. Aβ binding to neurons was quantified using the Image J 1.46r software by a modification of the method previously described [21]. Briefly, Aβ₄₂ and β-tubulin double-positive puncta were revealed by generating an Aβ₄₂/β-tubulin double-positive image, using the ‘and’ option of ‘image calculator’. A fixed threshold was set in the double-positive image, and the total co-localizing area was quantified using the ‘analyze particle’ function and normalized to total β-tubulin area in each field.

Electrophysiology

5000 HEK293T cells were seeded on 35-mm Petri dishes and cultured for two days in DMEM with 4.5 g/L glucose, without L-glutamine (VWR International PBI S.r.l., Milan, Italy), 10% fetal bovine serum, 1% penicillin-streptomycin 100X (Life Technologies, Milan, Italy), 1% UltraGlutamine1 (Lonza Group Ltd, Basel, Switzerland). HEK293T cells were perfused with solutions containing 10 μM Aβ₄₂ monomers, Aβ₄₂ oligomers or Aβ₄₂ oligomers pre-incubated for 30 min with 10 nM 15B3. Control cells were treated with the vehicle.

Membrane potential was monitored in single cells using the patch-clamp technique in configuration perforated-patch, current-clamp mode. In brief, patch-clamp pipettes (Garner Glass 7052) were made using a P97 Sutter Instruments puller (Novato, CA) and fire-polished to a tip diameter of 1–1.5 μm and 5–7 MΩ resistance. The Axopatch 200 B amplifier and pClamp 9 acquisition software and Clampfit 9 (both from Molecular Device, Novato, CA) were used to record and analyze cell membrane voltages. Experimental traces were digitized at 5 kHz and filtered at 1000 Hz. The bath solution contained (in mM) 136.5 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 5.5 glucose, 5.5 HEPES, pH 7.4. The perforated-patch clamp configuration was achieved by adding to the pipette solution (in mM) 135 KCl, 10 NaCl, 1 MgCl₂, 10 HEPES pH 7.2, and the antibiotic gramicidin (Sigma Aldrich) diluted to a final concentration of 7.5 μg/mL. This solution was used to fill the patch pipette allowing the pores in the membrane to open, to obtain electrical access to the cell after about 5–10 min. With this technique we can monitor the cell membrane potential for more than 30 min since cytoplasm dialysis is hampered by the sieve formed by the antibiotic apertures through the membrane which is permeable only to monovalent cations.

C. elegans experiments

Bristol N2 strain, from the Caenorhabditis elegans Genetic Center (CGC; University of Minnesota), was propagated at 20°C on solid nematode growth medium (NGM) seeded with OP50 Escherichia coli (from CGC) for food. To prepare age-synchronized animals, nematodes were transferred to fresh NGM plates on reaching maturity at three days of age and allowed to lay eggs overnight. Isolated hatchlings from the synchronized eggs (day 1) were cultured on fresh NGM plates at 20°C. For pumping-rate assays, nematodes (L3-L4 larval stage) were collected with M9 buffer, centrifuged, and washed twice with 5 mM PBS, pH 7.4 to eliminate bacteria. The worms were incubated with Aβ₄₂ without E. coli to avoid interference and bacteria-mediated peptide degradation. Worms (100 worms/100 μL) were incubated with 10 μM oligomeric Aβ₄₂ in 10 mM PBS (pH 7.4) alone or with the 15B3 antibody or control IgM. After 2 h, worms were transferred onto NGM plates seeded with OP50 E. coli. The pharyngeal pumping rate was scored 2 h later by counting the number of times the terminal bulb of the pharynx contracted in a 1-min interval (pumps/min).

Mouse studies

C57BL/6 mice were obtained from Charles River Laboratories (Calco, Italy). Procedures involving animals and their care were conducted in conformity with the institutional guidelines at the IRCCS – Mario Negri Institute for Pharmacological Research in compliance with national (Decreto Legislativo 4 marzo 2014, n.26) and international laws and policies (EEC Council Directive 2010/63/UE; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council (Eighth Edition) 2011). They were reviewed and approved by the Mario Negri Institute Animal Care and Use Committee which includes ad hoc members for ethical issues, and by the Italian Ministry of Health. Animal facilities meet international standards and are regularly checked by a certified veterinarian who is responsible for health monitoring, animal welfare supervision, experimental protocols, and review of procedures.

Intracerebroventricular (i.c.v.) cannulation was done as described [5]. Briefly, 10-week-old mice were anesthetized with Forane (Abbott Laboratories) and mounted on a stereotaxic apparatus (model 900, David Kopf Instruments, Tujunga, CA). A 7-mm-long guide cannula was implanted into the cerebral lateral ventricle (lateral ± 1.0 and dorsal-ventral –3.0 from the dura with an incisor bar at 0°) and secured to the skull with two stainless steel screws and dental cement. Mice were allowed 10–15 days to recover from surgery before the experiment.

Aβ₄₂ oligomers were prepared as described above, and diluted to a final concentration of 1 μM before i.c.v. microinfusion (7.5 μL/mouse). To test the effect of 15B3, mice were treated 5 min before the injection of Aβ₄₂ oligomers with 0.25 μg of antibody in 2 μL of PBS. The novel object recognition test (NORT) was run as described [5]. Briefly, mice are trained in an arena containing two objects that they can explore freely (familiarization phase). Twenty-four hours later, the mice are exposed to one familiar and one new object (test phase). Memory was expressed as a discrimination index, i.e., the time spent exploring the novel object minus the time spent exploring the familiar object, divided by the total time spent exploring both objects; the higher the discrimination index, the better the performance.

15B3 recognizes oligomeric but not monomeric forms of synthetic Aβ42

Aβ₄₂ was dissolved in PBS to a final concentration of 100 μM and incubated at 25°C in quiescent conditions. Aliquots were then taken at different times (from 0 to 72 h), diluted to 1 μM in PBS and flowed over SPR sensor chips on which the 15B3 antibody had been immobilized. Figure 1A shows that the binding signal on 15B3 was markedly affected by the length of incubation of Aβ₄₂. No binding signal was observed on injecting the freshly prepared solution (t = 0), whereas a marked increase was seen with solutions analyzed after 1, 2, and 5 h; with longer incubation (8, 24, and 72 h), the signal progressively declined (Fig. 1A,B). Injection of the 5-h Aβ₄₂ solution over immobilized control IgM resulted in a binding signal about 80% lower than that on immobilized 15B3 (Fig. 1C), supporting the specificity of the interaction. Results were confirmed by sandwich ELISA: 96-well microplates pre-coated with 15B3 were incubated with aliquots of Aβ₄₂ solution taken at 0–72 h. After washing, 15B3-captured Aβ₄₂ was detected using HRP-conjugated 4G8 antibody and chemiluminescence. The time-course of binding was similar to that obtained by SPR, with the highest signal at 5 h (Fig 1D).

We previously demonstrated [7] that freshly prepared Aβ₄₂ solution (t = 0) contained only monomers, whereas the solution incubated for 5 h contained a heterogeneous population of SDS-labile aggregates, including globular species and short protofibrils, with a main hydrodynamic diameter of 10–30 nm. The solution incubated for 24 h mainly contained larger, SDS-stable species [7]. Thus, 15B3 selectively recognized a specific population of soluble Aβ₄₂ oligomers, but not Aβ₄₂ monomers or higher-order aggregates.

The pseudo-irreversible binding observed around t = 5 h suggests multivalent interactions between different epitopes on a single oligomeric assembly and several immobilized 15B3 molecules. The affinity of the oligomers for 15B3 could not be determined because of the lack of information about their actual concentration, due to uncertainties about their precise molecular mass. However, based on previous analysis by size exclusion chromatography we can assume that after 5 h of incubation  40% of the monomers have assembled into oligomers with a mass range from 90 to 400 kDa [7, 22]; it follows that the concentration of oligomers is in the low nanomolar range (4–20 nM) and their affinity for 15B3 is exceptionally high (K_D < 1 nM).

The oligomeric population selectively recognized by 15B3 was also recognized by the anti-Aβ antibody 4G8, and by OC, a conformation-specific antibody believed to selectively target fibrillar oligomers [23], as shown by an SPR assay in which 4G8 or OC were flowed onto 15B3-captured oligomers (Fig. 2). This oligomeric population was not recognized by the other widely-used anti-oligomer antibody A11, neither in this SPR format (Fig. 2), nor when the Aβ₄₂ solution was flowed onto A11 immobilized on the sensor chip (data not shown). Dot-blot analysis confirmed the presence of species recognized by OC but not A11 in the 5-h Aβ₄₂ solution (data not shown).

15B3 inhibits Aβ₄₂ fibrillogenesis

Next we tested whether 15B3 binding to the soluble Aβ₄₂ assemblies formed early in the aggregation process affected subsequent fibril formation. A freshly prepared 4- μM solution of Aβ₄₂ was incubated with 20 μM thioflavinT (ThT) with or without 15B3 or control IgM, and the ThT fluorescence was monitored for 24 h [14]. Aβ₄₂ fibrillogenesis had a lag phase of 2 h, followed by very rapid growth, reaching a plateau after 5 h (Fig. 3A and B). Control IgM, up to 40 nM, did not significantly affect this process, whereas 15B3 shifted the curves in a dose-dependent manner, increasing the half-time of transition (Fig. 3C). There was some batch-to batch variability in the effects of 15B3, with batch 071114A about four times more potent than batches 061013 and 110531 (Fig. 3B). This might reflect subtle structural differences between antibodies of different batches, perhaps due to variable expression conditions, which might affect glycosylation and/or the intrinsic tendency of IgM to aggregate.

These data suggest that 15B3 binds to oligomeric intermediates on the pathway of Aβ₄₂ fibrillation. A similar effect on Aβ₄₂ fibrillogenesis was observed with N1, a physiological N-terminal cleavagefragment of PrP, which also selectively interacts with Aβ₄₂ oligomers [24].

15B3 inhibits the binding of Aβ₄₂ oligomers to recombinant PrP and to rat hippocampal neurons, and prevents oligomer-induced membrane depolarization

As a first step to assess the biological importance of the 15B3-Aβ₄₂ interaction, we investigated the antibody’s ability to counteract the binding of Aβ₄₂ oligomers to recombinant mouse PrP (rmPrP), since cellular PrP (PrP^C) may mediate oligomer-induced neurotoxic signaling [25]. Aβ₄₂ oligomers bound rmPrP immobilized on the sensor chip with high affinity, confirming previous results [5, 20] (Fig. 4). Preincubation of oligomers with 15B3 for 30 min resulted in dose-dependent reduction of binding (Fig. 4). 15B3 did not bind to immobilized rmPrP (data not shown).

Next, we analyzed the antibody’s ability to counteract the binding of Aβ₄₂ oligomers to rat hippocampal neurons [21]. Incubating neurons with monomeric Aβ₄₂ did not result in Aβ binding to the neuronal surface, as shown by the lack of staining with the anti-Aβ antibody 6E10 (Fig. 5A). In contrast, a strong immunopositive signal was seen on the surface of neurons incubated with the Aβ oligomer-containing solution (Fig. 5B). Preincubation of oligomers with 15B3 significantly reduced binding. This was clearly seen with batch 071114A (Fig. 5E); less so with batch 061013 (Fig. 5F), consistent with their different ability to inhibit Aβ₄₂ fibrillogenesis (Fig. 3). Control IgM (10 nM) had no effect on binding (Fig. 5F), and there was no 6E10-positive signal when neurons were incubated with 15B3 alone (data not shown). Experiments in which 15B3 was used to immunostain neurons that had been previously incubated with synthetic Aβ₄₂ oligomers were unrewarding. In fact, 15B3 could not even immunodetect pathological PrP in brain sections from prion diseased patients and mice ([10] and unpublished results), indicating that this antibody is not suitable for immunostaining.

Finally, we tested whether 15B3 protected cells from the effects of Aβ₄₂ oligomers on cell membrane potential. We used HEK293T cells, monitoring the cell membrane resting potential by a perforated-patch current-clamp technique [26]. HEK293T cells have a reduced set and a lower number of ion channels, and compensate small changes in membrane potential less efficiently than primary neurons, and are therefore more sensitive to the effects of Aβ₄₂. Acute exposure of cells to Aβ₄₂ oligomers, but not monomers, resulted in significant membrane depolarization, and this effect was completely abolished by 15B3 (Fig. 6).

15B3 prevents the toxic effects of Aβ₄₂ oligomers on C. elegans pharynx

We investigated the ability of 15B3 to counteract Aβ oligomer toxicity in vivo. First we used the invertebrate nematode C. elegans, whose pharyngeal behavior is sensitive to sublethal doses of chemical stressors. We previously reported that both rhythmic contraction and relaxation of the pharyngeal muscle in C. elegans, scored as “pumping rate”, were significantly impaired by feeding the nematodes synthetic Aβ₄₂ oligomers, but not monomers or fibrils [7]. Here we tested whether pre-incubation of 15B3 with Aβ₄₂ oligomers prevented this effect (Fig. 7). Consistent with previous observations, Aβ₄₂ oligomers significantly reduced the worm pumping rate, and this effect was dose-dependently antagonized by 15B3 but not by control IgM. 15B3 alone had no effect on the pumping rate (Fig. 7).

15B3 prevents the memory deficits induced by Aβ₄₂ oligomers in mice

We previously reported that synthetic Aβ₄₂ oligomers injected i.c.v. in C57BL/6 mice caused memory impairment in the novel-object recognition task [5]. This was not seen using either Aβ monomers or fibrils, indicating that Aβ oligomers are the molecular species responsible for the amnesic effect [5, 24].

We used this acute in vivo mouse model to evaluate whether 15B3 prevented the deleterious effects of Aβ₄₂ oligomers on memory. As expected, mice receiving 1 μM Aβ₄₂ oligomers, i.c.v., had a significantly lower discrimination index compared to mice injected with the vehicle solution (Fig. 8). No effect of Aβ₄₂ oligomers was seen in mice pre-treated i.c.v. with 15B3 (0.25 μg/2 μL, 5 min before oligomers) (Fig. 8).