Study design
The objective of our study was to determine whether USP14 plays a role in late-stage AD pathological characteristics and cognitive decline. We crossbred APP/PS1 transgenic mice, a widely used AD-like mouse model, with our newly created USP14+/- mice and aged the offspring to 12 months of age. The APP/PS1::USP14+/- mice were compared to APP/PS1, WT, and USP14+/- mice in terms of behavioral, neuropathological, and biochemical end points. Operators were blinded to mouse genotype for all outcome measures. We used power analysis for the Morris water test, the outcome measure with the highest variability, to compare cognitive behavior between 12-month-old APP/PS1::USP14+/- mice and APP/PS1 littermates and determined that we needed a minimum of 10 mice per group to achieve a statistical power of 80% for the α=0.05. To improve the rigor of the study, we included WT (n = 13), APP/PS1 (n = 9), APP/PS1::USP14+/- (n =8), and USP14+/- (n = 12) for behavioral tests and n = 3 to 6 mice per group for biochemical and immunohistochemical tests.
Generation of USP14+/- mice and APP/PS1 mice
The mice with heterozygous knockout of the Usp14 gene in the C57BL/6J inbred background (Usp14+/- mice) were generated through a contract to Shanghai Biomodel Organism Science &Technology Development Co., Ltd (Shanghai, China), using the CRISPR/Cas9 technology. In the knockout allele, the exon 5 of the Usp14 gene is deleted.
APPswe/PS1dE9 mice (AKA, APP/PS1 mice)[28], USP14+/-, and wild type (WT) C57BL/6J breeder mice were obtained from Shanghai Biomodel Organism Science &Technology Development Co., Ltd (Shanghai, China). WT, APP/PS1, USP14+/-, and APP/PS1::USP14+/- littermates resulting from crossbreeding between hemizygous APP/PS1 mice and USP14+/- mice were used in this study.
The protocol for the care and use of all animals in this study was in accordance with the Guangdong Animal Center for the ethical treatment of animals and approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University (SYXK2016-0168, Guangzhou, China). All mice were maintained in a temperature- and humidity-controlled room (22 ± 2 °C) and on a 12-h/12-h light/dark cycle. All animals had access to standard laboratory diet and drinking water ad libitum, and newborn mice were breastfed, and only males were used for this study to reduce gender-specific variability in the behavioral tests.
Genotype detection
Mouse tails were clipped and DNA was extracted with an extraction kit (Takara Bio Inc, catalog #9170A), and the genotype was detected using DNA polymerase chain reaction (PCR). The following primers were used: 1. APP/PS1,internal positive control: 5’-CTA GGC CAC AGA ATT GAA AGA TCT-3’ (Forward), 5’-GTA GGT GGA AAT TCT AGC ATC ATC C-3’(Reverse); PS1: 5’-AAT AGA GAA CGG CAG GAG CA-3’ (Forward), 5’- GCC ATG AGG GCA CTA ATC AT-3’ (Reverse). 2. USP14:5’-TCC CAA AAT AAA TGT AAA GTC AAT-3’ (Forward),5’-AAG GTG GGT AGA GAA AAT AAA AGT-3’ (Reverse).
Morris water maze
The spatial memory was blindly evaluated by the Morris water maze test [29, 30]. Before each experiment, the mice were brought to the site to allow them to be acclimatized. The temperature of the room and the water was kept at 24 ± 2°C. For spatial learning, the mice were trained to find a hidden platform for 6 consecutive days, 4 trials per day with a 20- to 30-second interval for each mouse from 3.00 to 8.00 p.m. On each trial, the mouse started from one of the four middle quadrants facing the wall of the pool and ended when the animal climbed on the platform. They were guided to the platform if they could not find the platform within 60 s. Through these training sessions, mice acquired spatial memory about the location of the safe platform. The swimming pathways and the latencies of the mice to find the hidden platform were recorded each day. The pathway and the length that the mouse passed through the previous platform quadrant were recorded by a video camera fixed to the ceiling of the room, 1.5 m from the water surface. The camera was connected to a digital-tracking device attached to an IBM computer loaded with the water maze software (Huaibei Zhenghua Biologic Apparatus Facilities). The spatial memory was tested 48 h later after the last training. When the platform was withdrawn, the path, distance or time was recorded; the longer a mouse stayed in the target quadrant where previously the platform had been located, the better it scored for the spatial memory (the distance and the time traveled in the target quadrant and the escape latency were used to indicate the score).
Westernblot analysis
Whole cell lysates and mouse cerebral cortex and hippocampal tissue homogenates were prepared in RIPA buffer (1×PBS,1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 10 mM β-glycerophosphate, 1mM sodiumorthovanadate,10 mM NaF,1mM phenylmethylsulfonylfluoride (PMSF), and1×Roche Complete Mini Protease Inhibitor Cocktail (Roche, Indianapolis,IN). SDS-PAGE, transferring,and immunodetection were performed as previously described [31]. In brief, equal amounts of total protein extracts were fractionated by12% SDS-PAGE and electrically transferred onto a polyvinylidenedifluoride (PVDF) membrane. Primary antibodies and appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were used to detect the designated proteins.The bound secondary antibodies on the PVDF membrane were reacted to the ECLdetection reagents (Santa Cruz, CA) and detected via exposing to X-ray films (Kodak,USA).
Enzymelinked immunosorbent assay (ELISA)
The hippocampus tissue was homogenized in RIPA buffer (Beyotime Biotechnology) containing a protease inhibitor cocktail (KeyGENBioTECH, catalog #KGP603), ultrasonicated, and centrifuged at 13000g for 10 min. The supernatant was collected and total protein was measured with a Micro BCA protein assay (Pierce). Aβ40 and Aβ42 were separately measured with ELISA Kits (R&D system) by following manufacturer’s instructions.
Quantitative reverse transcriptase (RT-) PCR (qRT-PCR)
Total RNA from brain tissues was isolated using the RNAiso Plus (Takara Bio Inc, catalog #9108). cDNA was prepared from the RNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara Bio Inc, catalog #RR047A). The real-time PCR amplification was performed using the SYBR Green method with SYBR Premix Ex TaqTM II (Takara Bio Inc, catalog #RR820A), and according to manufacturers’ instructions.
The following primers were used: APP: 5’- GCC AAC CAG CCA GTG ACC ATC-3 (forward), 5’-GCG ACG GTG TGC CAG TGA AG-3’ (reverse); PS2: 5’-CTG GCA ACG GAG ACT GGA ACAC-3’(forward), 5’-TGA ACA CAG CAA GCA GCA GGA G-3’ (reverse); USP14:5’- AAA CTT CTT CTA GTA TCC CAC C-3’ (forward); 5’- TGC TGA AGA TAC TGC CCT T -3’ (reverse); MAPT(Tau):5’-ACT CTG CTC CAA GAC CAA -3’ (forward), 5’- TCG GCT GTA ATT CCT TCT G -3’(reverse);β-actin:5’- GCT GTG CTA TGT TGC TCT A -3’ (forward), 5’- CGT TGC CAA TAG TGA TGA C -3’ (reverse). Results were normalized against the internal control using the ∆-∆CTmethod.
Immunohistochemistry
Mice were anesthetized and transcardially perfused rapidly with 50 ml normal saline solution, followed by fixation insituvia perfusion for 20 min at 4°C with Zamboni’s solution containing 4% paraformaldehyde, 15% saturated picric acid, and 24 mM NaH2PO4/ 126 mM Na2HPO4 (pH7.2). The brain was removed from the skull of the fixed animals and post-fixed in the same Zamboni’s solution for another 24 h at 4°C. Then coronal slices (8-10 µm) were cut with a Vibratome (Leica, VT1000S, Germany).
The immunohistochemical procedures were as follows [29]: Sections were permeabilized with 0.3% H2O2 in absolute methanol for 10 min to block endogenous peroxidase, and non-specificsites were blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature, and incubated with primary antibodies for 24 h at 4°C. After washing with PBS, sections were subsequently incubated with biotin-labeled secondary antibodies for 1 h at 37 °C. The immunoreaction was detected using horseradish peroxidase-labeled streptavidin for 1 h at 37 °C and visualized with the DAB tetrachloride system for brown color. A negative control for every antibody was used with PBS, then observed under a light microscopy (Olympus BX60, Tokyo, Japan) and photographed. The IODs and plaque burden in each image and were counted and measured using Image-Pro Plus v 6.0 software (Media Cybernetics Inc, Bethesda, MD, USA).
Gallyas-Braak silver staining
Silver staining was performed as previously reported [32]. In brief, brain sections (10 μm) were mounted on glass slides, hydrated through a series of alcohol solutions, then subject sequentially to the following treatments:1) 0.3% KMnO4 10 min, then washed to water clarification; 2) 1% C2H2O4• 2H2O 2 min, and La(NO3)3 solution (1.15 mM La(NO3)3•6H2O, 14.7 mM CH3COONa•3H2O) 1h, then washed 3×5min; 3) alkaline AgI(0.1 M NaOH, 0.06 M KI, 1% AgNO3 35ml) incubation 45minin at 37℃, avoided light, and the 1% acetic acid washing 3×1min; 4) the developer solution (Ⅰ:0.47 M Na2CO3;Ⅱ:25 mM NH4NO3, 11.77 mM AgNO3, 3.5mM H6O39SiW12; Ⅲ: 25 mM NH4NO3, 11.77 mM AgNO3, 3.5 mM H6O39SiW12, 6.1ml 40%HCHO; three solutions mixed equal parts, temporarily preparation and avoided light) incubated 30 min, then 1% acetic acid washed; 5) 0.5% AuCl3 30 s, and then washed 2×5min; 6) 1% NaS3O3•5H2O fixed 1min, and washed 10min. Then generally dehydrated, hyalinized, sealed, and then examined and photographed using a microscope (Olympus BX60, Tokyo, Japan) equipped with a digital camera.The IODs in each image were counted and measured using Image-Pro Plus v6.0 software (Media Cybernetics Inc,Bethesda, MD, USA).
Thioflavin-S staining
Brain sections (10 μm thickness) mounted on glassslides were hydrated through a series of alcohol solutions, soaked in 0.3% KMnO4 for 4 min, followed by a wash with 1% oxalicacid for 1 min [33]. Next, the brain sections were incubated with 0.05% aqueous Thioflavine-S (SIGMA, catalog#T1892) dissolved in 50% ethyl alcohol for 8 min at room temperature in dark, then washed in 50% ethyl alcohol twice and subsequently in three exchanges of distilled water. The slides were then incubated in a high concentration of phosphate buffer (411 mM NaCl, 8.1 mM KCl, 30 mM Na2HPO4, 5.2 mM KH2PO4, pH 7.2) at 4℃ for 30 min, washed, and finally sealed in 10% glycerin with coverslips. The stained sections were stored in a dark box at 4°C before they were observed with a fluorescence microscope (Leica, Germany) and photographed.The plaque burden in each image were counted and measured using Image-Pro Plus v6.0 software (Media Cybernetics Inc, Bethesda, MD, USA).
Statistical analysis
Water maze data are expressed as mean ± SEM, others are expressed as mean ± SD. GraphPad Prism 7.0 software (GraphPad Software) was used for statistical analysis. Differences between two groups were evaluated for statistical significance using two-tailed Student’s t-test. A difference among 3 or more groups, one-way ANOVA or when appropriate, 2-way ANOVA, followed by the Holm-Sidak test for pair-wise comparisons were performed. P value <0.05 was considered statistically significant.