Materials - GA (Copaxone®, 20 mg/mL) was acquired from Teva Pharmaceuticals (average molecular weight of 6.5 KD). HiLyte Fluor488 labelled Aβ1− 42 was acquired from AnaSpec, Inc. Monoclonal antibody W0-2 (IgG2a,k), which can specifically recognize Aβ N-terminal residues 5–8 region (10), was kindly provided by Dr. Qiaoxin Li. Alexa Fluor® 488 conjugated goat anti-mouse IgG2a secondary antibody was acquired from Thermo Fisher Scientific Australia. Auto-fluorescence control, isotype control, PerCP-conjugated anti-human CD45 antibody and APC conjugated anti-human CD14 antibody were purchased from BD Biosciences ANZ. RPE conjugated anti-human CD16 antibody was purchased from DAKO® Agilent Technologies Australia Pty Ltd.
Real time in vitro phagocytosis of beads assay
Human PBMCs (2 × 106/mL) were resuspended in 100 µL Hepes-buffered Na medium (145 mM NaCl, 5 mM KCl, 10 mM Hepes, 0.1% bovine serum album, 5 mM glucose, 0.1 mM CaCl2 and pH = 7.50). Cells were labelled with APC conjugated anti-human CD14 mAb at 4°C for 15 min. After staining, cells were washed once with Na medium and then resuspended in 1.0 mL Na medium. The phagocytosis assay was performed as previously described (8, 9). Briefly, all samples were kept at 37°C with constant stirring using a Time Zero module (Cytek Development Inc.). Cells were pre-incubated with 100 µg/mL CPX for 10 min or with 20 µM Cytochalasin D (Sigma-Aldrich, St Louis, MO) for 20 min at 37°C prior to the addition of 1.0 µm YG beads (Polysciences, Inc.). Cytochalasin D is a cell permeable fungal toxin that disrupts actin polymerization and inhibits phagocytosis, which is used to show non-specific binding by cells. The beads uptake was captured by a FACSCalibur flow cytometer (BD Bioscience). The linear mean channel of fluorescence intensity for each gated subpopulation over successive 10 s intervals was analysed by WinMDI software (version 2.9, by Joseph Trotter, The Scripps Research Institute, La Jolla, CA) and plotted against time. The beads uptake curve in the first 6 min was analysed. Fresh C57BL/6 mouse PBMCs (pooled blood from multiple animals) were labelled with APC conjugated anti-mouse CD11b mAb. The rest procedures are the same as above.
Microscale thermophoresis (MST) – MST was used to determine the interaction between GA and Aβ1−42. In brief, HiLyte Fluor488 labelled Aβ1−42 (80 nM, in PBS) was mixed 1:1 (volume) with serially diluted GA. Measurements were performed in standard treated capillaries on a Monolith NT.115 system using 95% LED and 40% IR-laser power. The IR-Laser was used to create a localized microscopic temperature gradient in the capillary. Simultaneously, local changes of fluorescence intensity due to the motion of Aβ in the glass capillaries were detected. The signal was recorded for IR-laser on and off stages with varying concentration of the GA. Changes of the thermophoretic property were observed as changes in fluorescence intensity.
Circular dichroism (CD) spectroscopy – CD is a useful technique for studying changes in the protein structure and protein–protein interactions in solution. CD in the far ultraviolet region (178–260 nm) arises from the amides of the protein backbone and is sensitive to the conformation of the protein. Molar ellipticity was acquired for Aβ1−42 alone, GA alone and mixture of both. Binding constants were determined by titration of Aβ by GA. Varied degrees of temperature on protein denaturation were employed to determine binding constants as well.
Western blotting – Samples containing Aβ and GA were mixed with loading buffer and reducing agent (DTT, 50 µM), followed by heating at 95 oC for 5 minutes. The mixtures were loaded to a 10-well pre-casted mini gel (NuPage). Electrophoresis was running with MES buffer at 100V for 50 minutes. Proteins were transferred to nitrocellulose membrane at 100V for 35 minutes. Immunodetection was performed using anti-Aβ antibody (clone W0-2) (10).
Aβ absorption on monocyte – Anti-coagulant human peripheral blood was treated for 15 minutes at 37°C with one of following treatments: 2.2 µM Aβ1−42 alone, 22 µM GA alone, or a mixture of both. After incubation, blood cells were washed twice, and red cells were lysed. Cells were stained with anti-Aβ mAb (clone W0-2) and the FITC conjugated secondary antibody, followed by extensive wash and CD14 and CD16 staining. Cells were analysed by flow cytometry.
Cell surface Aβ staining – Anti-Aβ antibody (clone W0-2) (20 µg/mL) was added into 100 µL whole blood. After a 15-min incubation at room temperature with gentle rocking, blood samples were washed twice with phosphate buffer and Alexa Fluor® 488 conjugated goat anti-mouse IgG2a secondary antibody was added at a final concentration of 15 µg/mL. After another 15-min incubation, red blood cells were lysed for 10 min using BD FACS Lysing Solution. Cells were then washed once with phosphate buffer and resuspended in 0.5 mL phosphate buffer. For further characterization of the positive cell populations, cells were washed again and stained with PE conjugated anti-human CD16 antibody, PerCP-conjugated anti-human CD45 antibody and APC conjugated anti-human CD14 antibody. Cells were washed and resuspended for FACS again. A tube of secondary antibody alone was used as control. All samples were tested together with an auto-fluorescence control, an isotype control and a secondary antibody alone control.
MTT cytotoxicity assay – SH-SY5Y cells were cultured in DMEM medium with 10% fetal bovine serum (FBS), 50 units/mL penicillin and 50 µg/mL streptomycin to full confluence. Cells (~ 10,000 in 100 µL) were then seeded to a 96-well plate and cultured for 48 hours. Media were discarded and replace by with 100 µL of Opti-MEM reduced serum medium free of phenol red or serum. Aβ1−42 (10 µM) was added to duplicate wells with escalating amount of GA by molar ratios: 1:0, 1:0.1, 1:0.5, 1:1, 1:3 and1:7. Vehicle and GA alone (10 µM) were used as controls. After 4 hours incubation, 10 µL of MTT (stock concentration of 12 mM) was added to culture media. A negative control was set using 10 µL of the MTT stock solution added to 100 µL of medium alone. Cells were then incubated at 37°C for another 4 hours. MTT formazan was dissolved in 50 µL DMSO and the absorbance was read at 540 nM.
Multi-electrode array (MEA) electrophysiology on LTP – An acute effect of GA on Aβ-induced LTP impairment in brain slices from naïve C57/BL mice and a chronic effect of GA on LTP in brain slices from 22-month old APP/PS1 mice with or without 28- day intracerebroventricular administration of GA were investigated. The procedure of recording LTP by MEA was described in previous studies (11, 12). Briefly, mice (C57BL/6J, aged from 12 to 15 weeks old or APP/PS1, aged 22-month-old after a 4-week treatment (icv) with or without GA), were anaesthetised with isoflurane and decapitated. Whole brains were quickly removed and place in ice-cold, oxygenated (95% O2, 5% CO2) cutting solution (composition in mmol·L− 1: 206 sucrose, 3 KCl, 0.5 CaCl2, 6 MgCl2–H2O, 1.25 NaH2PO4, 25 NaHCO3, and 10.6 D-glucose). A transverse hippocampal slice (350 µm) was prepared with a VT 1200S tissue slicer (Leica) and quickly transferred to 34°C artificial CSF (aCSF; composition in mmol·L− 1: 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.3 6 MgCl2–H2O, 1.25 NaH2PO4, 25 NaHCO3, and 10 D-glucose) for 30 min. After further recovery of 1-hr equilibrium in oxygenated aCSF at room temperature, the slices were transferred to a submersion recording chamber. An acute hippocampal slice was mounted on a MEA chip composed of 60 titanium electrodes (30-µm diameter) spaced 200 µm apart (60MEA200/30iR-Ti: MCS GnbH, Reutlingen, Germany). The slice was immobilized using a harp slice grid (ALA Scientific Instruments, New York, USA). The slice was continuously perfused with carbonated aCSF (3 ml·min− 1 at 32°C) during the whole recording session. Recordings were collected from the layer of stratus radiatum in CA1 as a major targeted dendritic synaptic site of Schaffer collateral projections from the CA1/CA3. Schaffer collaterals at CA1/CA3 were stimulated by injecting a biphasic current waveform (100 µs) through one selected electrode at 0.033 Hz. Care was taken to place the stimulating electrode in the same region from one slice to the other. Following a 20-min incubation period, slices were continuously stimulated with medium-strength stimuli. When stable evoked fEPSPs were detected (for at least 20 min), the stimulus threshold was determined. After recording at least 30 min of stable baseline of EPSPs, LTP was induced by applying three bursts of high frequency stimulus (HFS; 3 × 100 Hz, 500 ms width with 20-s intertrain interval) at the tested stimulation intensity and then fEPSPs were further recorded for another 30 min. Administration of an agent (either Aβ or copaxone or a mixture of both) was executed by 5 min’ (the 18th to 22nd min) perfusion of agent-contained aCSF. The peak-to-peak amplitude of field EPSPs (fEPSPs) was analyzed by using LTP-Analyzer (MCS GmbH, Reutlingen, Germany). LTP was expressed as the percentage increase of fEPSP 2 min after HFS (high frequency stimulation) over the baseline fEPSP.
Membrane fluidity test
Anti-coagulated peripheral blood (200 µL) was stained with PerCP-conjugated anti human CD14 and RPE-conjugated anti human CD16 for monocytes and incubated for 10 min at room temperature in the dark. The samples were then labelled with 5 µM of lipophilic probe TMA-DPH for 5 mins at 37oC. Following this, the samples were treated with 5 µM of Aβ1−42, 100 µg/mL of GA alone or 5 µM of Aβ1−42 and 100 µg/mL of GA or with no treatment as basal and incubated for 10 mins at 37oC. After incubation, the red blood cells were lysed for 15 mins at room temperature while protected from light. Then, the cells were washed twice and centrifuged at 1400 RPM for 3 mins. Finally, cells were resuspended with 1 mL of PB buffer and were examined on a Beckman flow cytometer (CytoFLEX LX). 50,000 events were acquired for each sample. Human monocyte population was identified by forward and side scatter and the expression of CD14.
Mouse study - APP/PS1 transgenic mouse is widely used as an animal model of AD. These mice start to accumulate Aβ in brain from 4 months; however, cognitive impairment only becomes evident from 10 months, and the lifespan is between 25–27 months. To better resemble the human AD, we used 22–24 months old female AD mice for this study. All animals underwent surgery to implant the mini-osmotic pump (ALZET, model 1004, 100µL reservoir volume, four weeks duration) plus ALZET Brain Infusion Kit 3. GA (20 mg/mL) (n = 9) or the control (isotonic saline containing 4% mannitol; n = 7) were loaded into minipump (100 µL each). Animals were anaesthetised with isoflurane inhalation and implanted with the mini-osmotic pump in the subcutaneous space in the back, penetrating 2.5 mm below the skull surface, which is appropriate for targeting the lateral ventricles in an adult mouse. The whole procedure followed the instruction as described previously (13). The reagents were expected to be released slowly within four weeks.
Mice underwent behavioural testing in the open field, elevated plus maze, Y maze, rotarod and social interaction over two consecutive weeks beginning at the 22th day after treatment. In the second week of behavioural testing includes rotarod and social interaction, three mice dropped off successively due to natural death or humane endpoint. Behavioural testing was conducted by an experimenter blinded to group assignment. Behaviour was recorded by an overhead camera, and behavioural metrices were objectively quantified using EthoVision tracking software (Noldus, Leesburg, VA, USA). To assess locomotion and anxiety-like behaviour, mice were tested in an open-field as previously described (14–16). Briefly, the mouse was placed in the center of the open-field and allowed to freely explore the arena for 5 minutes. The total distance moved, number of entries into the inner area of the maze, and time spent in the inner area of the maze were measured. Elevated plus maze (EPM) was used to evaluate anxiety-like behaviour, based on the tendency of rodents to avoid open spaces. During a 10 min period, mice were permitted free exploration of the apparatus, which consists of 2 open arms and 2 enclosed arms (San Diego Instruments, San Diego, USA). Distance moved and time spent in the open arms, as a percentage score, calculating by the time spent in the open arm/[time in open arm + time in closed arm], were examined. Spatial cognition was assessed in the Y maze as previously described. The Y-shaped apparatus consists of three enclosed arms of equal dimensions (San Diego Instruments, San Diego, USA), each with a different visual cue at the distal end. First, a 15-min habituation trial was conducted, in which the mouse was able to freely explore two arms while the third arm was blocked from access. After a 2-h interval, the third arm was unblocked, and a 5-min test trial was conducted, in which the mouse was able to freely explore all three arms. The number of entries into the novel and familiar arms and total distance travelled were determined. The three-chamber paradigm is a test to evaluate same-sex social affiliation and social recognition in mice. The task was performed as described previously (16). The apparatus has a central chamber and two outer chambers. Same-sex, age-matched, naive stimulus mice were restricted to rectangular metal cages fitted at one end of the outer chambers, such that experimental mice could only approach and initiate a social investigation from one side. Testing consisted of three consecutive 10-min stages: (1) habituation in the chamber, containing two empty cages; (2) a choice between an empty cage and a cage containing a stimulus mouse; and (3) a choice between a second ‘novel’ stimulus mouse and the first, now ‘familiar’ mouse. Stages 2 and 3 are measures of the subject’s preference for sociability and social recognition/social memory, respectively. Stimulus mice and their relative positioning (left versus right chambers) were randomised between subjects. Data were expressed as time spent in each of the outer chambers as a percentage of total time. The rotarod was used to assess motor function as previously described (15). The apparatus consisted of a rotating barrel divided by walls into four equal lanes (Harvard Apparatus, Holliston, TX, USA). Three trials were performed each day for two consecutive days (day 1 = training, day 2 = testing). For each trial, the mouse was placed on the rotating barrel, the speed was accelerated from 0.0027 to 0.27 g (4 to 40 RPM) at a rate of 0.00017 g/8 s, and the duration of time on the rotarod that the mouse was able achieve was recorded (maximum time of 5 minutes).
Following the completion of these behavioural tests, mice were euthanized, and the brain were collected for LTP measurement, immunohistochemical tests and Aβ quantification.
Histology and Immunohistochemistry
Mouse brains (GA, n = 4 and Controls, n = 4) were perfusion fixed with 4% paraformaldehyde and then placed in 10% neutral buffered formalin prior to standard histological processing for paraffin embedding and sectioning (5 µm). A survey section was deparaffinised and stained with haematoxylin and eosin.
Sections for Aβ (Dako MO872–clone 6F/3D) and microglia (Abcam ab178846) immunohistochemistry (IHC) were deparaffinised, rehydrated, and endogenous peroxidase blocked with 5% aqueous hydrogen peroxide (5 min). The sections for Aβ IHC were treated with 98–100% formic acid (Scharlau AC10852500) for 5 min, rinsed in water, and immersed in Tris buffer (0.5M pH 7.6) before a 1/100 dilution of MO872 was applied for 60 mins at room temperature. Sections for microglial IHC didn’t require antigen retrieval and the primary antibody was diluted 1 in 2000 in Tris and applied for 60 mins.
Positive labeling was detected with a peroxidase labeled streptavidin/biotin system (Dako K0675) with a diaminobenzidine chromogen. Sections were counterstained with Harris’s haematoxylin, dehydrated and coverslipped. Low and high magnification images were obtained with a Leica ICC50 HD camera on a Leica DM 750 binocular microscope. We then analyzed Aβ IHC with whole mount view (4X) by dissecting the brain section into hippocampal area (Suppl. Figure 3a) and cortical area (Suppl. Figure 3c). Aβ plaque structures were isolated and analyzed in Suppl. Figure 3b and d by method described elsewhere (17). We also analyzed microglial IHC sections (20X) based on method described previously (18). For Aβ IHC, the sections of two samples were not ideal in presentation of the hippocampal areas because of the positioning angle, and they were excluded from the quantitative analysis.
ELISA Aβ quantification
The Aβ levels were determined using the sandwich ELISA described previously (19). The assay was conducted in 384 well, high-binding polystyrene plate (Greiner). The plate was precoated with 25 µL of 10 µg/ml W0-2 monoclonal antibody (epitope Aβ 5–8) in 50 mM sodium carbonate, pH 9.6, at 4°C overnight. After washing with PBST (PBS containing 0.05% Tween), the plate was blocked with 0.5% (w/v) casein in PBS buffer, pH 7.4, to minimize nonspecific binding, and washed with PBS before addition of samples (in triplicate) and 1E8-biotin (epitope 17–22, final concentration 0.3 ng/ml) and incubated overnight at 4°C. The plate was washed with PBST and europium-labelled streptavidin (final concentration 1 nM) added for 1 hr at room temperature. After a final wash, the plate was developed with a commercial enhancement solution, and read using the Wallac Victor2 1420 Multilabel Plate Reader (PerkinElmer, Melbourne, Australia) with excitation at 340nm and emission at 613nm. The values obtained from the triplicated wells were used to calculate the Aβ concentration (expressed as ng/ml) based on the standard curve generated on each plate.
Rabbit study - Healthy female New Zealand White rabbits (2.7–4.6 kg) aged 12–15 weeks (n = 7) were purchased from a commercial rabbit breeding facility. Rabbits were randomized for various experimental purposes. They were housed in pairs or separately in a room kept at 21oC with a 12h light dark cycle. Rabbits were placed in a polycarbonate box and received local anaesthetic applied on ears subcutaneously (50/50 mix of 1% lignocaine + 1% ropivacaine with 30-gauge needle). YG beads (1.5 mL) was injected intravenously via marginal ear vein at a rate of 1.0 mL/ minute. After 5 min, GA at a dose of 2 mL/kg or PBS with 4% mannitol was injected into the marginal ear vein at a rate of 1 mL/ minute. Sample blood (200 µL) was collected from central ear artery of the other ear 5 min before bead injection as the baseline, followed by multiple collections at 5, 10, 15, 20, 30, 60 and 120 min after administration of GA or PBS. Blood samples were incubated with 4 mL of BD lysing solution for 15 min on ice, followed by washing with PBS and incubation with Alexa 647 conjugated anti rabbit CD14 mAb for 15 min. Cells were washed in PBS and the samples were analyzed by a BD FACSCalibur flow cytometry in gated CD14+ monocyte and CD14− neutrophil population.
Sheep study - In order to further verify the drug safety and obtain pharmacokinetic data, three adult sheep were selected for introductory experiments (36.5, 35.8 and 48.6 kg respectively). Ventricular cannulation (Intra-cerebroventricular surgery, ICV) was performed on sheep according to previously described methods (20). To test for GA drug safety, on the first day after the ICV operation, we injected 1 mL of carrier solution (4% mannitol in artificial cerebrospinal fluid, sterilized and filtered) from the ICV catheter at a rate of 0.5 mL/min (same rate for all the following injections). Cerebrospinal fluid (0.5 mL), blood (5 mL), and urine (5 mL) samples were collected to establish a baseline. GA was then injected once a week for 8 weeks at a dose of 10 mg in 0.5 mL. Heartbeat and breathing, anal temperature, forage consumption, drinking, defecation and urination were recorded daily.
To study the pharmacokinetics of GA, Alexa488 and Alexa647 conjugated GA was prepared using the conjugation kit (Thermo Fisher Scientific Australia) according to the manufacture’s instruction, the concentration was determined by Direct Detect (Merck), and the standard concentration curves were established with fluorescence spectrophotometry. Alexa488 or Alexa647 conjugated GA (10 mg) was injected via ICV. Cerebrospinal fluid (0.5 mL each time) was collected through intraventricular catheters, blood samples (5 mL each time) was collected through an external jugular vein cannulation, urine samples (5mL each time) was also collected, before GA injection and 0.5, 1, 2, 4, 8, 24, 48 and 72 hours after injection. The fluorescence intensity in each sample was measured and calculated with the standard curves.
Data analysis and statistics
Flow cytometry results were analyzed using FlowJo™ v10.8 Software (BD Life Sciences). Data analyzes and plotting were done using GraphPad Prism for Windows (Version 9.0, San Diego, California USA) or IBM SPSS Statistics for Windows (Version 28.0, Armonk, NY: IBM Corp.). For LTP assay, as there was no difference of basal fEPSPs between different treatment groups, a One-Way ANOVA was employed in comparison of LTP among different groups and the level of statistical significance was set up at p < 0.05. Two-way multivariate analyzes of variance (ANOVAs) were performed for social interaction and rotarod, with independent variables of treatment and chamber side (for social interaction) or treatment and day/training (for rotarod), and a (repeated) dependent variable of time, as appropriate. Triplicate values for each animal (for rotarod) were averaged before analyzing the data. Multiple comparisons were performed by Bonferroni-Šídák test and are annotated graphically where significant.