APPswe/PS1L166P mice with a C57/BL6 background were bred at the University animal facility. Transgenic APP/PS1 male mice were housed in the animal care facility for at least 2 weeks before starting the electrophysiological experiments. 7-8 month-old mice, weighing 28–35 g were used for the experiments. Animals were fed an autoclaved standard rodent diet (SDS, England; RMI-E), and maintained in a climate control animal facility with room temperature (22°C ±1), and relative humidity (46 ±5%). Mice had free access to food and water and were on a 12 h light/dark cycle.
Antibodies used for immunoblotting were as follows: CYFIP1 (1:1000, Upstate #07-531), FMRP (1:1000, Abcam #17722), 4E-BP2 (1:1000, Cell Signaling #2845), GAPDH (1:5000, Santa Cruz Biotechnology #32233), p-eIF4E (1:1000, Cell Signaling #9741), eIF4E (1:1000, Cell Signaling #9742), eIF4G (1:1000, Cell Signaling #2498).
In vivo electrophysiology in mice
Mice were anesthetized with urethane (injected i.p. 1.2 g/kg body weight), which was supplemented throughout surgery and recording as required. Mice were placed in a stereotaxic frame and body temperature was maintained at 37°C. In one hemisphere only, a bipolar stimulation electrode (NE-200, 0.5 mm tip separation, Rhodes Medical Instruments, Wood hills, CA) was positioned ipsilaterally onto the perforant path (3.8 mm posterior to bregma, 2.5 mm lateral to the midline, and 1.6 mm from the brain surface), while an insulated tungsten recording electrode (0.075 mm; A-M Systems) was positioned in the hilus of the DG (2 mm caudal to bregma, 1.5 mm lateral to the midline, and 1.5–1.7 mm from the brain surface). The recording electrode was lowered in 0.1 mm increments while monitoring changes in the stimulus-evoked waveform until the maximum, positive-going field excitatory postsynaptic potential (fEPSP) slope was obtained in the dentate gyrus. To generate input/output (I/O) curves, 5 stimulus intensities were used: 1) PS threshold, 2) 10% of maximum PS, 3) 30% of maximum PS, 4) 60% of maximum PS, and 5) maximum PS. The stimulus intensity ranged from approximately 80 µA (below PS threshold) to 400 µA (maximum PS). 4 responses were collected at each stimulus intensity and averaged. After generating an I/O curve, a stable 20 min baseline of evoked fEPSPs was recorded before delivery of high-frequency stimulation (HFS) to induce LTP. Stimulus pulses were biphasic, 0.1 ms pulse-width, delivered every 30 s (0.033 Hz) for baseline and post-HFS recording. The test pulse stimulation intensity was set to elicit a population spike amplitude of 30% of the maximum response. The HFS protocol consisted of 4 trains of stimuli with an interval of 10 s; each train consisted of 15 pulses at 200 Hz, at a stimulus intensity twice that used for test pulse. Evoked responses were recorded for 3 h post-HFS, and changes in the fEPSP slope were expressed in percent of baseline (average of 20 min baseline period). The maximum initial slope of the rising phase of the fEPSP was measured as an index of synaptic transmission efficacy. After completing the recording, mice were decapitated and the dentate gyri were micro-dissected and immediately frozen on dry ice for later use.
Tissue dissection and sample preparation
The ipsilateral (stimulated) and contralateral (control) DG were rapidly dissected on ice and homogenized in buffer containing 50 mM HEPEs, 100 mM NaCl, 1 mM EDTA, NP-40 0.5%, 1 mM dithiothreitol, 1 mM Na3VO4, 50 mM NaF, and 1× protease inhibitor cocktail from Roche #11836170001. Homogenization was performed manually with 10–12 gentle strokes in a tissue grinder and the homogenate was centrifuged 10 min at 14000 ×g at 4°C. Protein concentration was measured using BCA protein assay (Pierce, #23227). Homogenates were stored at -80°C until use.
Analysis of m7GTP binding proteins
For the m7GTP pull-down assay, 250-300 μg of protein lysate together with 30 μl of 7-methyl GTP-agarose beads (Jena bioscience #AC-141) were incubated for 90 min at 4°C. Beads were washed three times with m7GTP lysis buffer and bound proteins were separated to an SDS-PAGE (10% gels or 4–15% gradient gels). Immunoblotting was carried out as described below.
SDS–PAGE and immunoblotting
Samples from m7GTP pull-down assays and lysates were boiled at 70°C for 10 min in Laemmli sample buffer (Bio-Rad) and resolved in 10% SDS/PAGE gels. Proteins were transferred to nitrocellulose membranes (Biorad, #162-0112) which were then blocked with 5% BSA, probed with the respective antibodies, and developed using chemiluminescence reagents (Biorad, #1705061). The blots were scanned using Gel DOC XRS+ (BIO-RAD) and densitometric analyses were performed with Image J software (NIH, Bethesda, MD). In DG lysate (input) samples, densitometric values were expressed per unit of protein (GAPDH) applied to the gel lane. In the cap-pulldown analysis, protein expression was normalized to eIF4E. Values from the HFS-treated DG are expressed as percent change from the contralateral internal control DG of the same mouse.
Group values are reported as mean ±SEM. Statistical comparisons were calculated with the Two-way ANOVA with Bonferroni correction or multiple t-test using GraphPad Prism 8.02. The significance level was set at P <0.05.