Diffusible, highly bioactive oligomers represent a critical minority of soluble Aβ in Alzheimer’s disease brain.

Reagents and chemicals

Aβ1–40 and Aβ1–42 peptides were synthesized and purified using reverse-phase HPLC by Dr. James I. Elliott at the ERI Amyloid laboratory, Oxford, CT, USA. Peptide mass and purity (>99%) were confirmed by reverse-phase HPLC and electrospray/ion trap mass spectrometry. N-terminally extended (NTE) −31Aβ−40 was prepared and purified as described previously [ 47 ] and recombinant Aη-α (APP 505-612 ) was a gift from Drs. M. Willem and C. Haass (Ludwig-Maximillian University, Munich). Aη-α peptide was dissolved in 50 mM ammonium bicarbonate, pH 8.5, diluted to 10 ng/μl, aliquoted, and stored frozen at −80°C. Aβ and NTE-Aβ were dissolved in 50 mM Tris-HCl, pH 8.5, containing 7 M guanidium-HCl (GuHCl) and 5 mM ethylenediaminetetraacetic acid (EDTA) at a concentration of 1 mg/ml and incubated at room temperature (RT) overnight to disaggregate pre-existing seeds. Samples were then centrifuged for 30 minutes at 16,000 g and chromatographed on a Superdex 75 10/300 GL column eluted at 0.5 ml/min with 50 mM ammonium bicarbonate, pH 8.5. The concentration of the peak fraction for each peptide was determined from its absorbance at 275 nm. Peptide was then diluted to 10 ng/μl with the same buffer used for SEC, aliquoted and stored frozen at −80°C. When needed, an aliquot of a given peptide was thawed, used, and any remaining sample was discarded. Gel filtration standards were purchased from Bio-Rad (Hercules, CA). All other chemicals were of the highest purity available and unless indicated otherwise were obtained from Sigma-Aldrich (St. Louis, MO). For experiments involving Aβ peptides or brain extracts, protein Lo-Bind tubes (Eppendorf, Hamburg, Germany) were used.

Antibodies

The antibodies used in this study and their sources are described in Table 1 .

Preparation of human brain extracts

Human specimens were obtained from the Massachusetts ADRC Neuropathology Core, Massachusetts General Hospital and used in accordance with the Partners Institutional Review Board (Protocol: Walsh BWH 2011). Frozen temporal cortical tissues were obtained from a total of 10 cases, 9 of whom died with end-stage AD, and 1 subject who died free of AD ( Table 2 ). All AD cases met current post-mortem and clinical diagnostic criteria. Post-mortem intervals were less than 48 hours. Approximately 20 g of cortical gray matter was dissected from each case and this material was then sliced into ~2 g lots with a razor blade. Each lot was further cut into small chunks using a McIlwain tissue chopper (set at 0.5 mm). The diced tissue was gently mixed and divided in two. One half was used to prepare H extract and the other to prepare S extract ( Fig. 1 ). Both extracts were prepared using a buffer that we refer to as artificial cerebrospinal fluid base buffer (aCSF-B) (124 mM NaCl, 2.8 mM KCl, 1.25 mM NaH 2 PO 4 , 26 mM NaHCO 3 , pH 7.4). aCSF-B is the core buffer used in subsequent electrophysiology experiments. For preparation of brain extracts aCSF-B was supplemented with protease inhibitors (5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethyleneglycoltetraacetic acid, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 2 μg/ml pepstatin, 120 μg/ml pefabloc and 5 mM NaF). H extracts were prepared by homogenizing tissue in 5 volumes of ice-cold aCSF-B with 25 strokes of a Teflon-glass Dounce homogenizer (Fisher, Ottawa, Canada). Resulting 20% (w/v) homogenates were centrifuged at 200,000 g for 110 minutes and 4°C in a SW41 Ti rotor (Beckman Coulter, Fullerton, CA). The upper 80% of the supernatant was removed and designated as H extract . S extracts were prepared by incubating tissue in 5 volumes of ice-cold aCSF-B at 4°C for 30 minutes with gentle side-to-side mixing. Thereafter, this suspension was centrifuged at 2,000 g for 10 minutes and 4°C. The upper 90% of the supernatant was removed, centrifuged at 200,000 g for 110 minutes and 4°C in a SW41 Ti rotor. The resulting supernatant was removed and designated as S extract . S extracts necessarily include molecules derived from extracellular and intracellular compartments because prior to extraction, tissue underwent procedures that cause the rupture of cells (e.g. autolysis during the postmortem interval, freezing and thawing, dissecting, and slicing tissue). H2 extracts were prepared using the pellets generated when preparing S extracts . The 2,000 g and 200,000 g pellets were pooled and homogenized in 5 volumes of ice-cold aCSF-B, and centrifuged at 200,000 g for 110 minutes and 4°C. The upper 80% of supernatant was removed and designated as H2 extract . H , S and H2 extracts were then dialyzed against fresh aCSF-B. Fifty ml of extract was dialyzed (using Slide-A-Lyzer™ G2 Dialysis Cassettes, 2K MWCO, Fisher Scientific) against a 100-fold excess of fresh aCSF-B at 4°C, with buffer changed 3 times over a 72 hour period. Dialysis was used to remove small molecules such as excitatory amino acids and drugs that might interfere with our bioactivity assays, and the success of the process was confirmed by measuring the amount of glutamate in the final dialysate versus the starting extract. Thereafter, extracts were divided into 2 parts: 1 portion was immunodepleted (ID) of Aβ by 3 rounds of 12 hour incubations at 4°C with the anti-Aβ antibody, AW7, conjugated to Protein A Sepharose (PAS) beads [ 44 ]. The second portion was treated in an identical manner, but this time incubated with pre-immune serum conjugated to PAS beads. Samples were cleared of beads and 0.5 ml aliquots stored at −80°C until used for biochemical or bioactivity experiments. Samples were thawed once and used.

Measurement of soluble proteins in brain extracts

Total protein content in H , S and H2 extracts was measured using a Pierce BCA assay kit (ThermoFisher, Waltham, MA) in accord with suppliers’ instructions. Briefly, samples were diluted to 1:5 with aCSF-B and analyzed in triplicated versus bovine serum albumin (BSA) standards also prepared in aCSF-B with serial dilutions of BSA ranging from 0-2 mg/ml. To detect sAPP and BDNF, H , S and H2 extracts were mixed with equal volumes of 2× sample buffer and 10 μl of this was loaded in a single well and electrophoresed on either a pre-cast 16% polyacrylamide tris-tricine gel (for detection of BDNF) or a 10% polyacrylamide tris-glycine gel (for detection of sAPP) (Invitrogen, Carlsbad, CA). Gels were rinsed in transfer buffer (10% methanol, 0.192 M glycine, and 25 mM Tris) and transferred onto 0.2 μm nitrocellulose at 400 mA and 4°C for 2 hours. Membranes were blocked with 50% Odyssey blocking buffer in PBS for 1 hour at RT and then probed with appropriate antibodies. Monoclonal antibody 22C11 (Millipore, Billerica, MA) was used to detect sAPP and rabbit polyclonal antibody N-20 (Santa Cruz, Dallas, TX) was used to detect BDNF. Bands were visualized using a Li-COR Odyssey infrared imaging system (Li-COR, Lincoln, NE). The relative intensity of protein bands was determined and these values were used to estimate the percentage of sAPP and BDNF in S or H2 relative to H , i.e. S / H or H2/H x 100. Monomer-preferring MSD Aβ immunoassays The Aβx-40 and Aβx-42 assays preferentially detect Aβ monomers ending at Val40 and Ile 42, respectively. The x-40 assay uses monoclonal antibody (mAb) m266 (3 μg/ml), for capture and biotinylated 2G3 (0.2 μg/ml) for detection; the x-42 assay uses m266 (3 μg/ml) for capture and biotinylated 21F12 (0.4 μg/ml) for detection. Since incubation of samples with GuHCl dissociates soluble Aβ aggregates allowing increased detection of monomer by the Aβx-40 and Aβx-42 assays [ 29 ], samples were analyzed with and without pre-incubation in 5 M GuHCl. Briefly, 20 μl of extract was incubated with 50 μl of 7 M GuHCl at 4°C overnight. Thereafter samples were diluted 1:10 with assay diluent so that the final GuHCl concentration was 0.5 M. To match the buffer composition of standards with samples, monomeric stocks of Aβ1-40 and Aβ1-42 were prepared in Tris-buffered saline, pH 7.4 containing 0.5 M GuHCl, 0.05% Tween 20 and 1% Blocker A. Assays were performed using the Meso Scale Discovery (MSD) platform and reagents from Meso Scale (Rockville, MD). Samples, standards and blanks were loaded in triplicate and analyzed as described previously [ 28 ]. Oligomer-preferring MSD Aβ immunoassay This oAssay is >37,000-fold more selective for Aβ oligomers/soluble aggregates than Aβ monomer and uses amyloid-derived diffusible ligands as the calibrant [ 60 ]. The assay is performed essentially as described for the monomer-preferring assays, but employs the aggregate-preferring mAb, 1C22, for capture (3 μg/ml) and biotinylated 3D6 (0.4 μg/ml) for detection [ 28 , 60 ]. When Aβ aggregates are treated with GuHCl, the signal of this assays is greatly attenuated [ 29 ]. The percentage of different forms of Aβ in S or H2 relative to H was estimated using the values from the above 5 assays.

Culture of Chinese hamster ovary

(CHO) cell lines Media, fetal bovine serum (FBS), and media supplements were from Invitrogen (Carlsbad, CA). Naive, untransfected CHO cells were grown in Dulbecco’s modified Eagles medium (DMEM) containing 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. CHO cells stably transfected with human APP751 bearing the V717F mutation (which we refer to as 7PA2 cells) were grown in CHO medium plus G418 (200 μg/ml) [ 36 ]. Once cells reached 95-100% confluency, they were washed with 5 ml serum-free medium and incubated in 5 ml serum-free medium for an additional ~15 hours. Thereafter, medium was removed and centrifuged at 4°C and 200 g for 10 min. The upper 90% of the supernatant was transferred to a clean tube and centrifuged at 4°C and 3,000 g for a further 10 minutes. The upper 90% of the supernatant was removed and 5 mM EDTA was added to inhibit proteolysis. Finally, media was aliquoted into 2 ml lots and stored at −80°C. Immunoprecipitation/Western blot analysis of amyloid β-protein Extracts were first pre-cleared with PAS beads to minimize non-specific interactions in the subsequent IP. One ml aliquots of extracts were incubated with 15 μl PAS beads for 1 hour at 4°C with gentle shaking. PAS beads were removed by centrifugation (4,000 g for 5 minutes) and the supernatant divided into 0.5 ml aliquots. Each aliquot was incubated with 10 µl of AW7 and 15 μl PAS beads overnight at 4°C with gentle shaking. Aβ-antibody-PAS complexes were collected by centrifugation and washed as previously described [ 54 ]. The immunoprecipitated (IP’d) Aβ was eluted by boiling in 15 μl of 2× sample buffer (50 mM Tris, 2% w/v SDS, 12% v/v glycerol with 0.01% phenol red) and electrophoresed on hand poured, 15 well 16% polyacrylamide tris-tricine gels. Synthetic Aβ1-42 was run as a loading control and protein transferred onto 0.2 µm nitrocellulose at 400 mA and 4°C for 2 hours. Blots were microwaved in PBS and Aβ detected using the anti-Aβ40 and anti-Aβ42 antibodies, 2G3 and 21F12, and bands visualized using a Li-COR Odyssey infrared imaging system (Li-COR, Lincoln, NE). For certain experiments, the relative intensity of the ~4 kDa and ~7 kDa Aβ bands was determined and these values were used to estimate the percentage of species in S or H2 relative to H , i.e. S / H or H2 / H × 100. To determine if AW7 IP’d non-Aβ APP metabolites (e.g. sAPP, N-terminally extended Aβ or Aη peptides) from AD brain extracts, one milliliter aliquots of 7PA2 condition medium (7PA2-CM) or half milliliter aliquots of AD4 H extract were IP’d with either AW7 antiserum, or pre-immune serum (PI). The supernatant of AW7 IP’d 7PA2-CM was buffer-exchanged into 50 mM ammonium bicarbonate, pH 8.5, using a Zeba spin desalting column, lyophilized, and used for SDS-PAGE. Western blots were developed with 2E9, 6E10, or HJ2 plus 21F12 ( Table 1 ) and detected using ECL+ (Thermo Fisher Scientific, Rockford, IL).

Size exclusion chromatography

Samples were chromatographed on a Superdex 200 10/300 GL column eluted with 50 mM ammonium bicarbonate, pH 8.5 at 0.5 ml/minute. The column outlet was attached directly to a fraction collector and the elution of standards was monitored off line using a spectrophotometer. Each day prior to analyzing samples, the column was calibrated using Blue dextran and gel filtration standards. The peak fraction containing Blue dextran was designated as fraction zero. Two 0.5 ml aliquots of H or S extracts were removed from −80°C, thawed at room temperature for 20 minutes, pooled, vortexed and centrifuged at 12,000 rpm for 10 minutes. The upper 0.95 ml of sample was removed and loaded onto the SEC column and 0.6 ml fractions collected. To enable detection of Aβ of different aggregation states, fractions were lyophilized, then reconstituted in 60 μl of 5 M GuHCl and incubated at 4°C overnight. Thereafter, samples were diluted 1:10 with assay diluent and analyzed using the MSD-based Aβx-42 assay. To avoid cross-contamination of samples, no more than 3 brain samples were chromatographed on any given day, and in between samples 1 ml of 5 M GuHCl was loaded onto the column and eluted with at least 2 column volumes of buffer. At the end of each day, the column and collection tubing were thoroughly washed as described previously [ 44 ]. Experiments to isolate monomeric Aβ or NTE-Aβ peptides were performed using a Superdex 75 10/300 GL column connect to a BioRad BioLogic DuoFlow Chromatography System and eluted with 50 mM ammonium bicarbonate, pH 8.5 at 0.5 ml/minute. Production of induced neurons (iNs) from human induced pluripotent stem cells (iPSCs) Neurogenin 2 (Ngn2)-induced human neurons [ 63 ] were prepared as summarized in Supplementary Fig. 1 and as described previously [ 16 ]. Briefly, YZ1 iPSCs [ 62 ] were maintained in media containing DMEM/F12, Knockout Serum Replacement, pencillin/streptomycin/glutamine, MEM-NEAA, and 2-mercaptoethanol (all from Invitrogen, Carlsbad, CA) plus 10 μg/ml bFGF (Millipore, Billerica, MA). iPSCs were plated at a density of 95,000 cells/cm 2 for viral infection. Lentiviruses were obtained from Alstem with “ultrapure titres” and used at the following concentrations: pTet-O-NGN2-puro: 0.1 µl/50,000 cells; Tet-O-FUW-eGFP: 0.05 µl/50,000 cells; Fudelta GW-rtTA: 0.11 µl/50,000 cells. To induce Neurogenin 2 expression doxycycline was added on “iN day 1” ( Supplementary Fig. 1 ) at a concentration of 2 µg/ml. On iN day 2, puromycin was added at 10 mg/ml and maintained in the media at all time thereafter. On iN day 4, cells were plated at 5,000 cells/well on Matrigel (Corning, NY) coated Greiner 96 well microclear plates and maintained in media consisting of Neurobasal medium (Gibco), Glutamax, 20% Dextrose, MEM-NEAA and B27 with BDNF, CNTF, GDNF (PeprpTech, Rocky Hill, NJ) each at a concentration of 10 ng/ml. Prior studies indicated that neurite number and expression of neural markers reached near maximal levels by iN day 14 and that iNs were fully mature by iN day 21 [ 16 ]. To investigate the effects of AD brain extracts on neuritic integrity, cells were used at iN day 21. Addition of AD brain extract to induced neurons (iNs) and live-cell imaging Brain extracts were thawed on ice for 30-60 minutes, vortexed, centrifuged at 16,000 g for 2 minutes, and exchanged into neurobasal medium supplemented with B27/Glutamax using a HiTrap 5 ml desalting column (GE Healthcare, Milwaukee, WI). Briefly, two, 0.5 ml aliquots were pooled and applied to a desalting column using a 1 ml syringe at a flow rate of ~1 ml/min and eluted with iN culture medium. Ten, 0.5 ml fractions were collected. Prior studies indicated that fractions 4 and 5 contained the majority of eluted Aβ. Consequently, fractions 4 and 5 were pooled and used in subsequent iN experiments. A small portion (50 µl) of this material was also taken for Aβ analysis. Approximately 7 hours prior to exchanging AD brain extracts into culture medium, iN day 21 neurons ( Supplementary Fig. 1 ) were placed in an IncuCyte Zoom live-cell imaging instrument (Essen Bioscience, Ann Arbor, MI) and images collected every 2 hours for a total of 6 hours. This analysis was used to define neurite length prior to addition of brain extracts. Immediately after the acquisition of baseline images, half of the medium on iNs was removed (leaving ~100 µl) and 50 µl of buffer-exchanged extract or vehicle, plus 50 µl of fresh medium was added. Thereafter, images were collected from four fields per well every 2 hours for a total of 84 hours. Phase contrast images sets were analyzed using Incucyte Zoom 2016A Software (Essen Bioscience, Ann Arbor, MI). The ‘NeuroTrack’ analysis job was used to automatically define neurite processes and cell bodies [ 16 ]. Typical settings were: Segmentation Mode = Brightness; Segmentation Adjustment = 1.2; Cell body cluster filter = minimum 500 μm 2 ; Neurite Filtering = Best; Neurite sensitivity = 0.4; Neurite Width = 2 μm. Total neurite length (in mm) was quantified and normalized to the average value measured during the 6 hour period prior to sample addition. Immunocytochemical analysis of induced neurons (iNs) and confocal microscopy At the end of certain experiments, iNs were fixed, stained and used for confocal microscopy. Cells were fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA) and 4% sucrose at room temperature for 15 minutes, and then permeabilized with ice-cold methanol for 3 minutes. Cells were washed 3 times with PBS and then blocked using 5% (w/v) BSA in PBS containing 0.3% Triton X-100 and 0.02% sodium azide. Thereafter, iNs were incubated overnight with primary antibody (mouse anti-β-tubulin, Millipore, Billerica, MA; 2 μg/ml) at 4°C. Cells were again washed with PBS (x3) and then incubated for 1 hour at room temperature with fluorescence-conjugated secondary antibodies (AlexaFluor 546 goat anti-mouse; Invitrogen; at 2 μg/ml). Finally, iNs were incubated with DAPI (1 μg/ml in PBS, Invitrogen) for 15 minutes, washed 3 times with PBS and examined using a Zeiss LSM710 confocal microscope fitted with a 40x air objective (NA: 0.8). Images were captured in a Z-stack manner (15 stacks, interval 2 µm) and maximal pixel intensity projections were created with averaging of 2 frames set to 1024 × 1024 pixel resolution.

Mice

All animal procedures were performed in accordance with the National Institutes of Health Policy on the Use of Animals in Research and were approved by the Harvard Medical School Standing Committee on Animals. Wild type (WT) C57BL/6 mice were purchased from Jackson Labs (Bar Harbor, ME) and a small colony maintained in-house. Animals were housed in a room with a 12 hour light/dark circadian cycle with ad libitum access to food and water.

Brain slice preparation

Both male and female animals were used. At 2-3 months of age, mice were anaesthetized with isoflurane and decapitated. Brains were rapidly removed and immediately immersed in ice-cold (0-4°C) artificial cerebrospinal fluid (aCSF). The aCSF contained (in mM): 124 NaCl, 3 KCl, 2.4 CaCl 2 , 2 MgSO 4 ·7H 2 O, 1.25 NaH 2 PO 4 , 26 NaHCO 3 and 10 D-glucose, and was equilibrated with 95% O 2 and 5% CO 2 , pH 7.4, 310 mOsm. Coronal brain slices (350 µm) including hippocampus [ 54 ] were prepared using a Leica VT1000 S vibratome (Leica Biosystems Inc, Buffalo Grove, IL) and transferred to an interface chamber and incubated at 34 ± 5°C for 20 minutes and then kept at room temperature for 1 hour before recording. Long-term potentiation (LTP) recording Brain slices were transferred to a submerged recording chamber and perfused (10 ml/minute) with oxygenated (95% O 2 and 5% CO 2 ) aCSF 10 minutes before electrophysiological recordings. Brain slices were visualized using an infrared and differential interference contrast camera (IR-DIC camera, Hitachi, Japan) mounted on an upright Olympus microscope (Olympus, Tokyo, Japan). Recording electrodes were pulled from borosilicate glass capillaries (Sutter Instruments, Novato, CA) using a micropipette puller (Model P-97; Sutter Instruments, Novato, CA) with resistance ~2 MΩ when filled with aCSF. To induce field excitatory post-synaptic potentials (fEPSPs) in the hippocampal CA1, a tungsten wire stimulating electrode (FHC, Inc., Bowdoin, ME) was placed on the Schaffer collaterals of the CA3 and a recording electrode was placed at least 300 µm away on the striatum radiatum of the CA1. Test stimuli were delivered once every 20 seconds (0.05 Hz) and the stimulus intensity was adjusted to produce a baseline fEPSP of 30-40% of the maximal response of the initial slope of fEPSP. Thirty minutes following application of sample LTP was induced by theta burst stimulation (TBS). This involved 3 trains, each of 4 pulses delivered at 100 Hz, 10 times, with an interburst interval of 200 milliseconds with a 20 second interval between each train. Field potentials were recorded using a Multiclamp amplifier (Multiclamp 700B; Molecular Devices, Sunnyvale, CA) coupled to a Digidata 1440A digitizer. Signal was sampled at 10 kHz and filtered at 2 kHz and data were analyzed using Clampex 10 software (Molecular Devices, Sunnyvale, CA). Application of samples to LTP bath media Samples were stored frozen at −80°C in 0.5 ml aliquots and allowed to thaw at room temperature for 10 minutes and gently mixed by hand before using. For experiments when H extracts were diluted, stocks were thawed and diluted with aCSF-B immediately prior to use for LTP experiments. After a stable baseline had been achieved for at least 10 minutes, samples were added to the aCSF reservoir. The total volume in the reservoir, the recording chamber, the tubing and the pump was 9.5 ml, such that the effective dilution of each sample was 1:20. Thirty minutes after addition of sample, a TBS was delivered to induce LTP as described above. The experimenter was blinded to the identity of the H , S , ID, D, and aCSF samples, and samples were tested in an interleaved manner to avoid variances in animals or slice quality. Slices in each group came from different animals unless otherwise noted.

Statistical analysis

Electrophysiological data were analyzed offline by pCLAMP 10.2 (Molecular Devices, Sunnyvale, CA) and tested with One-way analysis of variance (ANOVA) with Bonferroni post-hoc tests or student t -tests. For live-cell imaging experiments, differences between groups were tested with ANOVA with Bonferroni post-hoc tests or student t -tests.

Supplementary Material Supplementary materials