Animals
Male B6C3-Tg (APPswe, PSEN1dE9)85Dbo/Mmjax transgenic hemizygous and wild-type (WT) littermates (The Jackson Laboratory, Bar Harbor, USA, https://www.jax.org/strain/004462) were maintained as a colony at the University of Otago. Animals were group-housed in standard caging until surgery at either 4 or 10 months of age. They were transferred to single housing at ~ 8 months of age to prevent injury from fighting between the males. Food and water were available ad libitum, and the cage contained one red plastic tube (approximately 5 cm in diameter, 10 cm long) and shredded paper bedding as standard housing. Animals were kept on a 12 h light:dark cycle (lights on at 7 am), and the room temperature was controlled via a thermostat set at 21 °C. All procedures were approved by the University of Otago Animal Ethics Committee and conducted in accordance with New Zealand Animal Welfare and Biosecurity Legislation.
Genotyping was carried out on tail tips which were lysed overnight at 55 °C in lysis buffer (100 mM Tris HCl pH 8.5, 5 mM EDTA, 0.2% (w/v) SDS, 20 mM NaCl) containing 20 μg/ml proteinase K. Isopropanol extracted DNA pellets were dissolved in TE buffer pH 8.0 (10 mM Tris, 1 mM EDTA). Polymerase chain reactions using two sets of primers that amplify the Psen transgene and mouse DNA as a positive control were carried out to distinguish between wild-type and transgenic animals. Primer sequences were obtained from the Jackson Laboratory (PsenTg_forward oIMR1644 AAT AGA GAA CGG CAG GAG CA, PsenTg_reverse oIMR1645 GCC ATG AGG GCA CTA ATC AT, control_forward oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT, control_reverse oIMR739 GTA GGT GGA AAT TCT AGC ATC ATC C). Agarose gel electrophoresis stained with ethidium bromide showed either one band that indicated a wildtype animal or two bands indicating a transgenic animal.
Lentivirus (LV)
Approval for the packaging and use of recombinant lentiviral vectors was obtained from the Environmental Protection Agency, NZ (GMD03091). The HIV-1 derived lentiviral plasmid, pCDH-EF1-MCS-T2A-copGFP (CD521A-1, System Biosciences, Palo Alto, CA) was modified to replace EF1 with the rat neuron-specific synapsin 1 promoter (Syn) [21] to drive neuronal expression of either copGFP (LV-control) or human sAPPα [22] and copGFP, separated by a T2A cleavage signal (LV-sAPPα). Vectors were packaged in HEK293FT cells using a second-generation packaging system [23]. Viral particles were pseudotyped with either the vesicular stomatitis virus (VSVg) envelope, which has tropism for a wide variety of cells, but has limited spread from injection sites [24, 25] or a chimeric rabies/VSVg (RabB19) envelope (Addgene #88865) containing the SADB19 (B19) extra-virion and transmembrane domains and the intra-virion domain of VSVg, which by contrast can undergo retrograde transport [26]. Average viral genome titres, determined by quantitative RT-PCR [23], were 2 × 1010 and 2 × 109 viral genomes/mL for VSVg and RabB19 pseudotyped LV, respectively.
Cell culture methods for detection of expressed sAPPa
Primary neuronal mouse cultures were prepared from postnatal day 2 C57BL/6 mouse pups. Animals were deeply anaesthetized with pentobarbital (150 mg/kg, s.c.) and decapitated. After removing meninges and cerebellum from the brains, tissue was diced finely and then digested for 15 min at 37 °C on a MACS-Mix (Miltenyi Biotec, DE) in Leibovitz’s L-15 medium (Life Technologies, NZ) supplemented with 20 mM D-(+)-glucose (Sigma Aldrich, NZ), 0.8 mM kynurenic acid (Sigma Aldrich, NZ), 0.05 mM D(−)-2amino-5-phosphovaleric acid (AP5; Sigma Aldrich, NZ), 50 U/mL penicillin, 0.05 mg/mL streptomycin (penicillin-streptomycin; Life Technologies, NZ), 5.5 mM L-cysteine HCl (Sigma Aldrich, NZ), 12 U/ml Papain (Worthington Biochemical Corporation, NJ, US), 1 U/ml DNaseI (Life Technologies, NZ), 1.1 mM EDTA, 0.067 mM beta-mercaptoethanol, and 2% (v/v) B27 (Life Technologies, NZ). The enzymatic digest was stopped by blocking for 10 min at 37 °C on a MACS-Mix (Miltenyi Biotec, DE) in Leibovitz’s L-15 medium supplemented with 20 mM D-(+)-glucose, 0.8 mM kynurenic acid, 0.05 mM AP5, 50 U/mL penicillin, 0.05 mg/mL streptomycin, 10 mg/mL BSA and 10 mg/mL ovomucoid (Sigma Aldrich, NZ). Tissue was then triturated in OptiMEM supplemented with 20 mM D-(+)-glucose, 0.4 mM kynurenic acid, 0.025 mM AP5, 10 mg/mL BSA and 2% B27, passed through a 100 μm cell strainer and cells pelleted by centrifugation. The cell pellet was resuspended in culture media (Neurobasal A (Life Technologies, NZ) supplemented with 35 mM D-(+)-glucose, 0.4 mM L-glutamine (Life Technologies, NZ), penicillin (50 U/mL) and streptomycin (50 mg/mL), and 2% B27). Cells were plated at a density of 200,000 cells / well of a 24-well plate containing poly-L-lysine hydrochloride (Sigma Aldrich, NZ) coated coverslips. Cells were maintained in culture media in a 37 °C/5% CO2 incubator, with half of the volume replaced with fresh media every 3 days [27]. Cultures were transduced at 6 days in vitro (DIV) by adding 4 μl/well lentivirus expressing Syn.sAPPα-T2A-copGFP or Syn.T2A-copGFP, respectively. For immunocytochemistry, cells were fixed in 4% paraformaldehyde at 10 DIV and then stained with a MAP2 antibody (Millipore Cat# MAB3418 RRID:AB 11212326, 1:1000)/goat-anti-mouse Alexa488 (Cat# A-11001 RRID:AB_2534069, Life Technologies, NZ; 1:1000), and 4′, 6-diamindino-2phenylindole (DAPI; Life Technologies, NZ). For western blotting, media was replaced with culture media without B27 the day after transduction, and collected at 10 DIV.
Stereotaxic surgery
At 4 or 10 months of age (prevention and rescue studies, respectively), animals were anaesthetised with a subcutaneous injection of ketamine/domitor/atropine (75/1/0.05 mg/kg body weight), and placed into a stereotaxic frame (Kopf Instruments; California, USA). Vectors were bilaterally injected through a 33 ga needle into the hippocampus using 2 μL of viral preparation per hemisphere at a rate of 150 nL/min. Four injection sites per hippocampus were used to optimize virus spread. Stereotaxic coordinates from bregma were (in mm): AP -1.8, ML ±1.2, DV -1.25 and − 1.95; and AP -2.5; ML ±1.8, DV -1.25 and − 1.95. The needle was left in place for 3 min after each injection before moving to the next site.
For surgeries at 4 months of age, a total of 25 WT animals were injected with LV-control, 16 Tg animals injected with LV-control, and 28 Tg animals injected with LV-sAPPα. For surgeries at 10 months of age, 26 mice were injected for electrophysiological analysis of LTP (9 WT with LV-control, 7 Tg with LV-control, and 10 Tg with LV- sAPPα).
Behavioural testing
Behavioural testing commenced at 12 months of age, eight months after surgery in the 4 month age group. All behavioural testing and data analysis were conducted by an experimenter blind to the treatment conditions.
Open field
The open field test was conducted in a 40 × 40 × 25 cm opaque white plastic box. The mouse was placed in the middle of the box and its behaviour observed and recorded for 5 min with a ceiling-mounted video camera linked to a computer running Ethovision XT7 software. The centre zone was defined as the 24 cm × 24 cm area in the middle of the open field and the percentage time spent in the periphery or the centre of the field was measured. At the end of the trial, the mouse was removed from the box, fecal boli were removed and the box cleaned with 10% ethanol.
Morris water maze
The Morris watermaze testing was performed in a white plastic circular pool with a diameter of 100 cm and filled with water (20–22 °C) until 9 cm from the top. A small circular transparent Perspex platform (diameter 6 cm) stood 0.7 cm under the surface of the water and 21.5 cm from the pool wall. Prominent visible spatial cues with dissimilar features were located around the room at different heights. Performance was recorded using a ceiling mounted camera linked to the Ethovision XT7 program. Day 1 consisted of habituation by placing the mouse into the pool without the platform for 1 min. Days 2 and 3 comprised the cued learning phase, during which a visible flag was attached to the submerged platform (SE quadrant) and the mouse learned to seek out the platform. Each mouse underwent 6 trials/day with a maximum time in the pool of 60 s/trial and an inter-trial interval of 3 min. When the mouse reached the platform, it was allowed to remain on it for 15 s, and if the mouse did not reach the platform within 60 s, it was then gently placed on the platform and left there for 15 s.
The spatial reference memory acquisition phase was conducted on days 4–9, with 6 trials a day for 6 days, an inter-trial interval of 3 min, and the platform maintained in a fixed position different from during cued learning. The same platform was used for all sessions and each trial began from a different pseudo-randomly chosen start position with the mouse facing the wall. The mice were each allowed a maximum of 60 s in the pool, and if the mice did not arrive at the platform within the 60 s, they were then picked up and placed on the platform and allowed to remain there for 15 s. Total distance travelled (path length) and proximity data (calculated as the average distance from the platform during a trial and considered a sensitive measure of spatial learning [28, 29]), were measured.
Probe trials to test spatial reference memory were conducted just prior to training on the fourth day of reference memory acquisition (probe trial 1), and then 24 h after the last day of acquisition (probe trial 2). The mouse was placed in the pool for 60 s without the platform present and the number of platform crossings and proximity data were measured.
Immediately following probe trial 2, three days of spatial working memory testing were conducted. The experimental protocols were the same as for the reference memory acquisition testing except that the platform location was different for each day, although fixed for each day. The first day of testing was used for familiarizing the mice with the working memory task, and data were collected and analysed for the next two days of testing.
Object recognition
Following a rest day, the mice were re-habituated to the open field box for 5 min. The object recognition task began the next day and consisted of placing two distinctly different objects in the centre of two adjacent quadrants of the box. The objects used (consisting of a plastic cube [4 × 4 × 4 cm], a cylinder [4 × 4 cm diameter], and a pyramid [4 × 4 × 4 cm] had 1.5 cm holes drilled into its sides in order to increase exploration [30]. The following day, one of the objects was replaced with a novel object with a different shape in order to test novel object recognition; 24 h later, the familiar object was moved to another quadrant of the box to test novel object recognition. The objects replaced and displaced were counterbalanced between mice. Each mouse was placed in the box and allowed to explore for 5 min. Exploring was defined as the mouse’s direct interaction with the object, such as nose and paw touching. Mice that did not achieve a total of 10 s of exploration within the given 5 min were excluded from the study. One WT-control, one Tg-control, and three Tg-sAPPα mice were excluded from the study based on these criteria. The trials were recorded by an overhead camera and mouse behaviour observed and analysed by the experimenter off-line. All objects and the exploration box were cleaned with 10% (v/v) ethanol solution between trials. For each object recognition task, the amount of time the animal spent exploring each object was measured. The data were then converted into a discrimination ratio, defined as:
$$ \frac{\mathrm{exploration}\ \mathrm{time}\ \mathrm{of}\ \mathrm{novel}\ \mathrm{object}\ \left(\mathrm{or}\ \mathrm{location}\right)-\mathrm{exploration}\ \mathrm{time}\ \mathrm{of}\ \mathrm{familiar}\ \mathrm{object}\ \left(\mathrm{or}\ \mathrm{location}\right)}{\mathrm{total}\ \mathrm{exploration}\ \mathrm{time}} $$
Post-mortem tissue preparation
Beginning at least one week after the end of the behavioral testing, animals were deeply anaesthetized with pentobarbital (200 mg/kg, s.c.) and a transcardial perfusion was conducted with an ice-cold sucrose dissection solution (mM: 210 sucrose, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 3 MgCl2, 20 D-glucose) which had been bubbled with carbogen (95% O2–5% CO2). Following removal of the brain, one hemisphere was assigned for hippocampal slice electrophysiology and the other hemisphere for post-mortem analyses including western blots, ELISAs and histochemistry. The assigned hemisphere for each analysis alternated between left and right for successive mice.
Extracellular electrophysiology
After removing the frontal cortex and cerebellum, the selected hemisphere was sectioned transversely into 400 μm coronal slices using a Leica vibrotome (VT 1000). Slices were transferred to a Millipore cell culture insert (Millicell®, Millipore, MA, USA) housed in a custom built incubation chamber containing artificial cerebrospinal fluid (ACSF, mM: 124 NaCl, 3.2 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2.5 CaCl2, 1.3 MgCl2, 10 D-glucose) bubbled with carbogen. The slices were subsequently incubated at interface for 30 min at 32 °C and then at room temperature for at least 90 min. After this recovery period, slices were transferred to a recording chamber where they were gradually warmed to 32.5 °C while superfused (2 mL/min) with oxygenated (with carbogen) and humidified ACSF.
All recordings were made by an experimenter blind to the genotype and treatment condition of the mice. Field potentials were evoked using stimulating electrodes made from 50 μm Teflon-insulated tungsten monopolar electrodes placed in either the alveus or stratum radiatum and driven by custom constant-current stimulators controlled by custom Labview software. Evoked potentials were recorded using glass micropipettes (1.5–2.5 MΩ filled with ACSF), amplified (× 1000), filtered (0.3 Hz-3 kHz) and stored for later analysis using custom software. Population spikes were recorded in the stratum pyramidale in order to assess cell excitability across stimuli ranging from 10 to 200 μA (average of 3 responses at each stimulus intensity) to generate an input-output (I-O) curve, and then to assess recurrent inhibition by paired-pulse stimulation (PPI), where stimulation was first applied to the alveus to antidromically activate CA1 pyramidal cell axons (antidromic spike 75% of maximum amplitude), and then the stratum radiatum to evoke orthodromic population spikes (50% of maximum amplitude). Interpulse intervals ranged from 20 to 200 ms, with two pairs of stimuli at each pairing interval, followed by one orthodromic stimulus alone in association with each interval. PPI was expressed as the average of the two orthodromic responses for each pair at each interval divided by the average of all the orthodromic-only responses.
After PPI assessment, the recording electrode was moved to stratum radiatum where field excitatory postsynaptic potentials (fEPSPs) were recorded. Basal synaptic transmission was assessed by the input-output (I-O) measurements of fEPSPs by applying stimulation at increasing intensities as described above. Presynaptic paired-pulse facilitation (PPF) was tested by giving the slice three consecutive stimulations at interpulse intervals ranging from 20 to 200 ms. PPF was expressed as a ratio and was calculated as pulse 2 amplitude/pulse 1 amplitude. In LTP experiments, the stimulus current was set at a value that yielded half maximum fEPSP slope and the slice was stimulated every 30 s while a 30 min baseline was recorded. LTP was induced by giving either two (prevention study) or three (rescue study) theta-burst stimulation protocols (TBS) spaced 30 s apart. Each TBS protocol comprised 10 bursts at 5 Hz, with 5 pulses at 100 Hz per burst, at baseline stimulus intensity. After TBS, responses were recorded for a further 120 min. The initial slopes of the fEPSPs were measured, and each response expressed as a percentage change from baseline, which was defined as the average of the last 20 responses before TBS.
Histochemical analysis
Coronal sections (40 μm) from frozen tissue were mounted on slides and allowed to dry overnight. Congo-red was used to stain the sections to reveal amyloid plaques, with nuclei labelled with DAPI. Congo-red staining and DAPI were visualised on a Zeiss AX10 fluorescence microscope, attached to a Jenoptic camera and computer, and the percentage area covered by plaques was analysed using ImageJ. In short, images were converted to 8 bit, a threshold value was determined and maintained for all images, and the percentage area covered by plaques was calculated using the ImageJ algorithm.
Western blots
The hippocampi not used for electrophysiology were snap-frozen on dry ice and stored at − 80 °C until protein extraction. Protein was extracted in solubilisation buffer (5 mM phosphate buffer pH 7.4, 0.32 M sucrose, 0.5 mM phenylmethylsulfonyl fluoride [PMSF in ethanol], 1 mM EGTA, 1 mM EDTA, and a protease inhibitor (cOmplete Ultra Mini Tablet, Roche)) without detergent, homogenized by pestle 30× and supernatant collected by two centrifugation steps at 14,000 g for 10 min and 30 min respectively at 4 °C. The resulting supernatant was identified as the soluble fraction. The resulting pellets were solubilized in a second buffer containing Triton-X and SDS (EGTA 1 mM, EDTA 1 mM, PMSF 0.5 mM, cOmplete protease inhibitor, Triton-X (1% v/v), sodium dodecyl sulphate (0.1% w/v) in phosphate buffered saline pH 7.4) and proteins solubilised by probe sonication (10 pulses at 1 s each; Qsonica, CT, USA). The resultant fraction was identified as the insoluble fraction. A DC protein assay (Bio-Rad) was use to quantify protein concentrations in both fractions.
Protein samples were separated on 9 or 12% (w/v) bis-acrylamide gels before transferring to a nitrocellulose membrane. Blots were incubated in Odyssey blocking buffer (LI-COR) at room temperature for 1 h. The primary antibody (microglia: Iba-1, WAKO 019–19,741, RRID:AB_839504; astrocytes: GFAP, Abcam-AB10062, RRID:AB_296804; presynaptic boutons: synaptophysin, Abcam-AB32127, RRID:AB_2286946; postsynaptic density: PSD-95, BD Transduction 610,496, RRID:AB_397862) or tubulin (Abcam-AB4074, RRID:AB_2288001) was prepared in phosphate buffered saline (PBS)-tween, 0.1% (w/v) BSA and 0.1% (v/v) NGS, overnight at 4 °C. The secondary antibody was either IRDye goat anti-rabbit680 or IRDye goat anti-mouse800 (LI-COR (1:10,000) in PBS/Tween), 1 h at room temperature. Blots were imaged on a LI-COR Odyssey imaging system, quantified using Image Studio 4 (LI-COR) after normalising to a loading control protein (tubulin).
Detection of sAPPα in the cell culture media was achieved by western blotting. Media was initially concentrated by ammonium sulfate precipitation (at 75% saturation). Proteins were then separated on a 10% (w/v) SDS-PAGE gel and transferred to a nitrocellulose membrane (100 V, 1 h). Blocking overnight in 1% (w/v) milk powder-PBS tween was followed by incubation for 2 h at room temperature with an N-terminal APP antibody (Cat# A8967 RRID:AB_258427, Sigma Aldrich, NZ; 1:1000), diluted in blocking solution (1% milk powder-PBS tween).. After three washes in PBS-0.3% Tween-20 (PBS-T), anti-rabbit-HRP secondary antibody (Cat# NA934, RRID:AB_772206, GE Healthcare Life Sciences) was applied for 2 h at room temperature (1:10,000 in PBS-T). Unbound secondary antibody was removed with three PBS-T washes and the blot was developed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) and imaged using a Fuji LAS-3000 ECL imaging system.
Enzyme-linked immunosorbent assay (ELISA)
Aβ and sAPPα concentrations of the hippocampal samples were measured using four ELISA kits: Human amyloid β (1–42) Assay Kit (IBL, Hokkaido, Japan, 27,711), human amyloid β (1–40) Assay Kit (IBL, 27,713), human sAPPα high sensitive ELISA (IBL, JP27734), and Mouse/Rat sAPPα (highly sensitive) ELISA (IBL, JP27419). The procedures were performed according to the kit instructions. ELISA for mouse and human sAPPα were performed on the soluble fraction (as prepared for western blotting), and ELISA for human Aβ (1–42) and (1–40) were performed in both the soluble and insoluble fractions. Despite its ability to detect recombinant human sAPPα samples, the human sAPPα kit was not able to detect either native or virus-mediated sAPPα expression in the Tg mice, and thus we could not determine degree of up-regulation of sAPPα levels in the tissue. Therefore copGFP expression was used as the marker of successful transduction in the hippocampus.
Statistical analysis
Behavioural and electrophysiological statistical data were calculated in Microsoft Excel and SPSS v21 (IBM), and differences between groups were compared using one-way analysis of variance (ANOVA) or a mixed model two-way ANOVA with repeated measures on one factor with Lower-Bound corrected values. Post-hoc tests were conducted using Tukey’s test, with significance set at p < 0.05. All group data are presented as mean ± SEM. Planned comparisons were conducted using Students t-tests comparing WT-control group with Tg-control group to examine genotype, and Tg-control group with Tg-sAPPα group to determine treatment effect. T-tests were conducted using SPSS version 21 software.