Human subjects and autopsy of brain tissues
Postmortem frozen brain tissues were obtained from the Oregon Brain Bank at Oregon Health and Science University (OHSU; Portland, OR, USA). Donor subjects of either sex were enrolled and clinically evaluated in studies at the National Institutes of Health (NIH)-sponsored Layton Aging and AD Center (ADC) at OHSU, in accordance with protocols approved by the OHSU Institutional Review Board (IRB). Informed consent was obtained from all participants prior to their enrolment in brain aging studies at the ADC; each subject received annual neurological and neuropsychological evaluations, with a Clinical Dementia Rating assigned by an experienced clinician. A neuropathological assessment was performed at autopsy in compliance with IRB-approved protocols. A neuropathologist scored autopsy brain tissue for Aβ plaques and neurofibrillary tangles according to standardized CERAD (Consortium to Establish a Registry for AD) criteria and Braak staging (22). The tissues used in this study were from subjects classified as controls because they had normal cognitive examination results (Mini-Mental State Examination scores 0-30) (23). Donor subject samples were de-identified by the ADC prior to shipment to the University of Texas Medical Branch (UTMB), so no approval was required from the UTMB IRB under CFR §46.101(a)(1). The cases used in this study are described in Supplementary Table 1 (Additional file 1).
Wild-type 11- to 13-weeks-old male and female mice (C57BL/6J Mus musculus-Cat# JAX:000664, RRID: IMSR JAX:000664) and 9 male 3xTgAD mice (B6.Cg-Tg(APPSwe,tauP301L)1Lfa Psen1tm1Mpm/2J Mus musculus-Cat# JAX: 033930) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Health care was provided by the animal care specialists under supervision of the facility manager. Animal colony care and maintenance were provided on a daily basis to ensure a safe, healthy environment. Each animal was used under a protocol approved by UTMB’s Institutional Animal Care and Use Committee, ensuring that the animals experienced the minimal amount of pain/discomfort. All animals were housed under USDA standards (12:12-h light/dark cycle, ad libitum food and water) at the UTMB vivarium.
The synaptosomal fraction containing both pre- and postsynaptic components was isolated by using a well-established method developed in our laboratory (24–26). Briefly, we lysed snap frozen hippocampus (HP) and frontal cortex (FC) tissue from mouse and cognitively intact elderly human brains using SynPER lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) with 1% protease and phosphatase cocktail inhibitors. The brain homogenates were centrifuged at 1200 × g for 10 min at 4°C. The supernatants (containing the synaptosomes) were collected and centrifuged at 15,000 × g for 20 min at 4°C. The synaptosomal pellets were resuspended in HEPES-buffered Krebs-like (HBK) buffer (143.3 mM NaCl, 4.75 mM KCl, 1.2 mM MgSO4·7 H2O, 1.2 mM CaCl2, 20.1 mM HEPES, 0.1 mM NaH2PO4, and 10.3 mM D-glucose, pH 7.4). Finally, 0.5% of Pluronic F-68 non-ionic surfactant (cat# 24040-032, lot# 2275337; Thermo Fisher Scientific) was added to prevent synaptosome aggregation as previously described (27). The quality and concentration (synaptosomes/µl) of isolated synaptosomes was routinely verified by flow cytometry and electron microscopy as we previously reported (24). Sample processing for the ultrastructure, flow cytometry, and protein analyses are described in details in the Supplementary Methods (Additional file 1).
Human Aβ1–42 peptide was purchased from Department of Biophysics and Biochemistry, Harvard University (Cambridge, MA, USA), and AβO were prepared from lyophilized synthetic Aβ aliquots as previously described (28). Briefly, 0.3 mg of lyophilized Aβ were dissolved in 200 µl of 1,1,1,3,3,3-hexafluoro-2-propanol (HFP). Then, 700 µl of double-deionized water was added, and the sample was magnetically stirred in a fume hood for 48 h at room temperature (RT). A cap with four holes was placed on the tube containing the sample to allow HFP evaporation. The obtained AβO were aliquoted, frozen at −80°C, and used within 3 months of preparation. Flow cytometry analysis of AβO binding to synaptosomes was performed using AβO spiked with HyLite Fluor 647-tagged Aβ (cat# AS-64161; AnaSpec Inc., Fremont, CA, USA). These AβO (AβO647) were prepared by adding 7 µl of tagged Aβ to the HFP-Aβ mixture prior to AβO formation. Oligomeric preparation quality was checked by western blot analysis using the AβO-specific 6E10 antibody (cat# 803002; BioLegend, San Diego, Ca, USA). AβO have been well characterized by our group and others (10,29–32).
Prepared recombinant TauO were provided by Dr. Rakez Kayed’s laboratory. They were produced and characterized following established and published protocols (33,34) and labeled (TauO488) as previously described (35–37). Briefly, 1 mg of Alexa Fluor™ 488 NHS Ester Succinimidyl Ester (cat# A20000, Thermo Fisher Scientific) was dissolved in 0.1 M sodium bicarbonate to a final concentration 1 mg/ml, pH 8.3. The dye was then incubated with TauO in a 1:4 (w/w) ratio, rotating overnight at 4°C on an orbital shaker. The following day, the solution was centrifuged (30 min, 15,000 × g) using 10-kDa Amicon Ultra-0.5 ml Centrifugal Filter Units (cat# UFC501024; EMD Millipore, Burlington, MA, USA) to remove unbound dye. TauO were then washed with 1× phosphate-buffered saline (PBS) until the flow-through solution was clear. The filter compartment was then flipped and centrifuged to collect the concentrate (2 min, 1000 × g). Oligomer concentrations were then quantified with the Pierce™ BCA Protein Assay Kit (cat# 23227, Thermo Fisher Scientific) and used for flow cytometry and immunofluorescence analyses.
AβO and TauO binding challenge to synaptosomes
Synaptosomes were treated with AβO and/or TauO for binding challenges, and the binding percentages were evaluated with flow cytometry. We pooled together an equal number of synaptosomes isolated from each sample. We incubated 2 million of synaptosomes for 1 h at RT without oligomers (control) as well as with AβO tagged with HyLite Fluor 647 or/and TauO tagged with Alexa Fluor™ 488 NHS Ester (Thermo Fisher Scientific) at concentrations of 0-0.5-1-2.5-5-10 µM. Synaptosomes were then pelleted, washed three times with HBK buffer, and resuspended in HBK. Oligomer fluorescence positivity was acquired by a Guava EasyCyte 8 flow cytometer (EMD Millipore) and analyzed using Incyte software (EMD Millipore).
AβO and Tau oligomers binding challenge to mouse brain slices
For this set of experiments, 3- to 4-month-old C57BL/6J mice were euthanized with deep isoflurane anesthesia followed by cervical dislocation, and the brains were immediately collected and sliced using a Compresstome VF-300 (Precisionary Instruments, Greenville, NC, USA) in N-methyl-D-glucamine-artificial cerebrospinal fluid (NMDG-aCSF) buffer (93 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 M sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 10 mM MagSO4·7H20, 0.5 mM CaCl2 2H2O, and 12 mM N-acetyl L-Cysteine) to obtain 350-µm horizontal brain sections. Slices were allowed to recover for 30 min in NMDG-aCSF-NaCl buffer at 33°C. Slices were then maintained at RT in a modified HEPES holding-aCSF solution (92 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 2 mM MgSO4 7H20, 2 mM CaCl2 2H2O, 12 N-Acetyl L-Cysteine). For oligomer challenges, the slices were incubated for 1 h at RT with AβO and/or TauO at concentrations of 0-0.05-0.5-1-2.5 µM. After treatment, synaptosomes were isolated, pelleted, washed three times with HBK buffer, and resuspended in HBK buffer. Oligomer fluorescence positivity of synaptosomes was measured as described above.
Proteinase K digestion
Following the challenge experiments, synaptosomes were digested with 1 mg/ml of proteinase K (PK; cat# 70663-4, lot# 3018798; EMD Millipore) for 30 min at 37°C (1 mg of PK is the equivalent of 30 mAU, where AU is an Anson unit that represents the amount of enzyme that liberates 1.0 µmol [181 µg] of tyrosine from casein per min at pH 7.5 at 37°C). The remaining oligomer binding positivity was measured by a Guava EasyCyte flow cytometer (EMD Millipore) and analyzed using Incyte software (EMD Millipore). For pretreatment experiments, synaptosomes were digested with 1 mg/ml of PK (cat# 70663-4, lot# 3018798; EMD Millipore) for 30 min at 37°C prior to challenge with labeled AβO and TauO. Binding was measured as for the other experiments.
Synaptosomes were treated with AβO and/or TauO for binding challenges, and the binding percentages were evaluated by western blotting analyses. We pooled together an equal number of synaptosomes isolated from each sample. We incubated 2 million of synaptosomes for 1 h at RT without oligomers (control) as well as with AβO and TauO (prepared as above) at the concentrations of 0-2.5-10 µM. Synaptosomes were washed three times with HBK buffer, and the pellets were lysed with 1× radioimmunoprecipitation assay buffer (RIPA buffer) with 1% protease and phosphatase cocktail inhibitors. Tricine sample buffer (Thermo Fisher Scientific) was added to the total proteins derived from AβO-treated synaptosomes to a final concentration of 1×. Then, proteins were loaded in a 16% Novex Tricine gels (Thermo Fisher Scientific) followed by 45 minutes transfer to Amersham Protran nitrocellulose transfer membranes (GE Healthcare-Life Sciences, Chicago, IL, USA) at 85 V at 4°C. The use of tricine sample buffer and gel is well recommended for the detection of low molecular weight proteins. Lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific) was added to the total proteins derived from TauO-treated synaptosomes to a final concentration of 1×. Then, proteins were loaded in a 12% NuPAGE Bis-Tris gels (Thermo Fisher Scientific) followed by 1 h transfer to Amersham Protran nitrocellulose transfer membranes (GE Healthcare-Life Sciences) at 95 V at 4°C. The membranes were blocked using Odyssey blocking buffer (LI-COR, Lincoln, NE, USA) for 1 h at RT and incubated at 4°C overnight with the anti-Aβ antibody (6E10, cat# 803002; RRID:AB_2564654; 1:1000 dilution; BioLegend) or the anti-tau antibody (Tau5; cat# 806402; RRID:AB_2564706; 1:1000 dilution; BioLegend) and 1 h at RT with the anti-synaptophysin (SYPH) antibody (cat# ab8049; RRID:AB_2198854; 1:10,000 diluted; Abcam, Cambridge, UK). All primary antibodies were prepared in a 1:1 solution of 1× Tris-buffered saline solution with Tween (TBST) and Odyssey blocking buffer. After incubation, the membranes were washed three times with 1× TBST (10 min each) and incubated 1 h with LI-COR secondary antibodies diluted at 1:10,000 in 1× TBST-Odyssey blocking buffer at RT. The membranes were again washed three times for 10 min each. Western blots were imaged using a LI-COR Odyssey infrared imaging system, application software version 3.0.30. The density of each immunoreactive bands were measured using ImageJ software (https://imagej.nih.gov/ij, NIH, Bethesda, MD, USA).
Primary neuron isolation
Primary cortical neuronal cultures were prepared and maintained as described previously (37). Briefly, cortical neurons were isolated from C57BL/6 mice during embryonic days 16-18 by gentle trituration by a fire-polished glass pasteur pipet with Accutase® solution (cat# A6964, Sigma, St. Louis, MO, USA). Dissociated cells were plated at a density of 5 × 105 cells/ml in Ibidi µ-Slide 8 Well Glass Bottom (cat# 80827; Ibidi GmbH, Martinsried, Germany) containing high-glucose Dulbecco’s Modified Eagle’s Medium (cat# 10–013-CV; Corning, Corning, NY, USA) with 2% B-27 Plus supplement (cat# A3582801; Gibco/Thermo Fisher Scientific), 10,000 U/ml penicillin, 10,000 µg/ml streptomycin, and 25 µg/ml amphotericin B (cat# 15240062, Gibco/Thermo Fisher Scientific). After 2 h, plating medium was removed from ls and replenished with neurobasal medium (cat# 12348017, Gibco/Thermo Fisher Scientific) plus 2% B-27 Plus, 0.5 mM GlutaMax (cat# 35050-061, Gibco/Thermo Fisher Scientific), 10,000 U/ml penicillin, 10,000 µg/ml streptomycin, and 25 µg/ml amphotericin B supplement. Half of the medium was changed every 3-5 days. Cells on days 7-10 in vitro were used for all experiments.
Primary neuron treatment and immunofluorescence
Cultured neurons were exposed to AβO647 and TauO488 at concentrations of 2.5 µM at 37°C for 30 min. After treatment, oligomer-containing media were removed, and cells were washed three times with 1× PBS (5 min each). Cells were fixed with 300 µl of 4% paraformaldehyde (PFA)/PBS for 15 min at RT. Cells were then permeabilized in 300 µl of 0.25% Triton X-100 in PBS (PBST) for 10 min at RT and then washed three times in 1× PBS (5 min each). Blocking was done in 300 µl of 5% normal goat serum/5% bovine serum albumin (BSA) in PBST for 1.5-2 h. Primary antibodies were diluted 1:500 in 5% BSA/PBST for overnight incubation at 4°C (microtubule-associated protein 2 [MAP2] antibody, cat# MAP2; Aves Labs, Tigard, OR, USA; postsynaptic density-95 [PSD95] antibody, cat# ab13552, RRID:AB 300453, Abcam). After primary antibody incubation, the cells were washed three times in PBST (10 min each). Secondary antibodies from Thermo Fisher Scientific were diluted in 5% BSA/PBST for 2 h at RT (Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594, cat# A11007, RRID:AB_141374; 1:400 diluted; Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 405, cat# A48260; 1:250 diluted).
Intracerebroventricular (ICV) injections
16 months old 3xTgAD mice were anesthetized with isoflurane and subjected to ICV injections using the freehand injection method previously described (38,39). Briefly, a 29-gauge needle, firmly held with hemostatic forceps to leave 4.5 mm of the needle tip exposed, was connected to a 25 µl Hamilton syringe via 0.38 mm polyethylene tubing. Infusions were performed at the rate of 3 µl/min for a total volume of 3 µl, using an electronic programmable microinfuser (Harvard Apparatus). Mice were ICV injected with 3ul of 0.55µM of either AβO or TauO. After ICV injection, the needle was left in place for 2 minutes, while the mouse was allowed to recover lying on a heated pad under warm light. 24 hours after ICV injection of oligomers or PBS, mice were euthanized and the brains were removed, dissected, snap frozen on dry ice, and stored at − 80°C. Then, we isolated synaptosomes from hippocampus and frontal cortex and subjected the protein extracts to western blot analysis to evaluate the effects of the ICV injected AβO and TauO on endogenous TauO and AβO respectively in comparison with the control mice (ICV with PBS).
Fluorescence-assisted single synaptosome long-term potentiation
Fluorescence-assisted single synaptosome long-term potentiation (FASS-LTP) is a chemically induced LTP technique (cLTP) focused on the insertion of glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the postsynaptic surface, which is an essential event for synaptic transmission potentiation. FASS-LTP identifies potentiated synapses by tracking GluA1 and neurexin-1β (Nrx1β) surface expression in size-gated, glycine-activated synaptosomes. FASS-LTP experiments were conducted as previously described (40–42). Briefly, 5 × 106 synaptosomes were suspended in separate tubes containing different solutions. Tube E contained 200 µl of extracellular solution (20 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 15 mM glucose, and 15mM HEPES, pH 7.4), tube B contained 200 µl of basal solution (20 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 15 mM glucose, and 15 mM HEPES, pH 7.4), and tube C contained 200 µl of cLTP solution (150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 30 mM glucose, and 10 mM HEPES, pH 7.4). Synaptosomes in extracellular solution were used to determine the basal levels of potentiated synaptosomes. All tubes were incubated at RT on a slowly oscillating shaker to thaw the frozen samples. For stimulation, 20 µl of 5 mM glycine (N-methyl D-aspartate receptor [NMDAR] co-agonist) was added to the tube C freshly supplemented with 0.001 mM strychnine and 0.02 mM bicuculline methiodide. Equivalent amounts of extracellular solution were added to control tubes E and B. Following stimulation, synaptosomes in tube C were depolarized with 100 µl of KCl solution (50 mM NaCl, 100 mM KCl, 2 mM CaCl2, 30 mM glucose, 10 mM HEPES, 0.5 mM glycine, 0.001 mM strychnine, 0.02 mM bicuculline) and incubated at 37°C for 30 min. This step is based on the principle that high KCl concentrations depolarize synaptosomes to release endogenous glutamate, which further activates synaptic NMDARs in conjunction with the co-agonist glycine. Equivalent amounts of extracellular solution were added to tubes E and B and incubated along with tube C for 30 min at 37°C. The contents in tubes E, B, and C were transferred to 15-ml centrifuge tubes. Then, 0.5 ml of ice-cold 0.1mM EDTA-PBS solution and 4 ml of 5% blocking buffer (5% fetal bovine serum in PBS) were added to tubes E, B, and C to stop the reaction. Tubes were kept on ice and centrifuged at 2500 × g for 5 min at 4°C, and the supernatant was discarded. The translocated AMPARs are then captured by adding to the tubes B and C, 2.5 µg/ml of the primary antibodies specific for the extracellular epitopes GluR1 (anti-GluR1 antibody, cat# ABN241, RRID:AB_2721164, EMD Millipore) and Nrx1β (cat#75-216, RRID:AB_2155531; Antibodies Incorporated, Davis, CA, USA) prepared in blocking solution. After incubation with the primary antibodies and subsequent washes with 1× PBS, the samples were centrifuged at 2500 × g for 5 min at 4°C. Pellets were resuspended in 100 µl of secondary antibody solution (Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647, cat#A-21236, RRID:AB_2535805; Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, AlexaFluor 488, cat#A-11034, RRID:AB_2576217; both from Thermo Fisher Scientific). After 45 min of incubation at 37°C, synaptosomes were washed twice with 1× PBS and then centrifuged at 2500 × g for 5 min. Endogenous/nonspecific background fluorescence for each marker was determined using secondary antibody staining only in tube B containing synaptosomes maintained in external solution (37°C, 45 min); no differences in background fluorescence were found between tubes B and C. After the second wash, pellets were resuspended in 400 µl of 2% PFA in PBS and maintained at 4°C in the dark. Samples were acquired a Guava EasyCyte flow cytometer (EMD Millipore) and analyzed using Incyte software (EMD Millipore).
Statistical analyses were performed using GraphPad Prism version 9.1.0 software. T test two-tailed, one-way ANOVA with Dunnett’s multiple comparison test, or two-way ANOVA with Tukey’s multiple comparison test were used to detect significant differences between groups. Data were then expressed as the mean ± SD, and for all statistical analyses p = 0.05 was considered as statistically significant.