P301S(PS19) transgenic mice (B6;C3-Tg(Prnp-MAPT*P301S)PS19Vle/J, stock number: 008169) were obtained from the Jackson Lab . Both male and female PS19 mice and age-matched non-transgenic wild-type (WT) littermates at 7 months of age were used in the present study. 4-Nitrophenyl-4-[bis (1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate (JZL184, provided by the NIH Mental Health Institute Chemical Synthesis and Drug Supply Program), a potent and selective inhibitor of MAGL, was dissolved in the vehicle containing Tween-80 (10%), DMSO (10%), and saline (80%). Animals were treated with either vehicle or JZL184 (10 mg/kg, i.p.) three times per week starting at 5 months of age for 8 weeks, as described previously [16, 19]. All the observations and assessments in mice were made at 7 months of age.
All the experiments were performed in compliance with the US Department of Health and Human Services Guide for the Care and Use of Laboratory Animals.
The care and use of the animals reported in this study were approved by the Institutional Animal Care and Use Committee of University of Texas Health San Antonio.
The experiments, whenever possible, were performed in a blinded fashion.
Levels of IL-1β and TNFα in hippocampal tissues of PS19 TG mice that received vehicle control or JZL184 were detected using a colorimetric IL1β and TNFα ELISA kits (IL-1β: Cat# RAB0275, Sigma-Aldrich, and TNFα: Cat# MBS825075, MyBioSource) according to the instructions provided by the manufacturers.
Western blot assay was conducted to determine expression of GluA1, GluA2, GluN1, GluN2A, and GluN2B, tau-5, AT8, p-tauT181, p-GSK3β, p35/25, PPARγ, PSD95, synaptophysin (Syn) in the hippocampus from animals that received vehicle or JZL184. Hippocampal tissue was extracted and immediately homogenized in RIPA lysis buffer and protease inhibitors, and incubated on ice for 30 min, then centrifuged for 10 min at 10,000 rpm at 4oC. Supernatants were fractionated on 4–15% SDS-PAGE gels and transferred onto PVDF membranes (Bio-Rad). The membrane was incubated with anti-GluA1 (1:1,000, Abcam, Cat# AB31232), GluA2 (1:1,000, Abcam, Cat# AB133477), GluN1 (1:1,000, Abcam, Cat# AB109182), GluN2A (1:1,000, LSBio, Cat# LS-B7707), GluN2B (1:1,000, Abcam, Cat# AB65783), tau-5 (1:1,000, ThermoFisher, Cat# AHB0042), AT8 (1:200, ThermoFisher, Cat# MN1020), p-tauT181 (1:1,000, Cell Signaling Technology, Cat# 12885), p-GSK3β (1:1,000, Cell Signaling Technology, Cat# 9323), p35/25 (1:1,000, Cell Signaling Technology, Cat# 2680), PPARγ (1:1,000, Abcam, Cat# AB27649), Syn (1:500, Abcam, Cat# AB8049), and PSD95 (1:1,000, Abcam, Cat# AB2723) at 4oC overnight. The blots were washed and incubated with a secondary antibody (goat anti-rabbit 1:2,000, Cell Signaling Technology, Cat# 7074) at room temperature for 1 hr. Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences, UK). The densities of specific bands were quantified by densitometry using FUJIFILM Multi Gauge software (version 3.0). Band densities were quantified and converted to the total amount of protein loaded in each well as determined by mouse anti β-actin (1:2,000, Santa Cruz Biotechnology, Cat# SC-47778) as described previously [16, 19, 28, 29].
Immunohistochemical analyses were performed to determine astrocytic and microglial markers (GFAP and Iba1) in coronal brain sections, as described previously [16, 19, 30]. Animals were anesthetized with ketamine/Xylazine (200/10 mg/kg) and subsequently transcardially perfused with PBS followed by 4% paraformaldehyde in phosphate buffer. The brains were quickly removed from the skulls and fixed in 4% paraformaldehyde overnight, and then transferred into the PBS containing 30% sucrose until sinking to the bottom of the small glass jars. Cryostat sectioning was made on a freezing Vibratome at 40 µm and series sections (every 10th section from each animals) were collected in 0.1M phosphate buffer. Free floating sections were immunostained using specific antibodies for GFAP (1:500, Sigma-Aldrich, Cat# G3893) an Iba1 (1:500, Sigma-Aldrich, Cat# MABN92) followed by incubation with the corresponding fluorescent-labeled secondary antibody. 4′-6-Diamidino-2-phenylindole (DAPI), a fluorescent stain that binds strongly to DNA, was used it to detect cell nuclei in the sections. The sections were then mounted on slides for immunofluorescence detection using a Zeiss deconvolution microscope. The immunoreactivity (in arbitrary densitometric units) of GFAP and Iba1 in the cortex and hippocampus in each image was analyzed and quantified using SlideBook 6.0, as described previously [16, 19, 27, 30].
Novel object recognition
The novel object recognition (NOR) test was performed as described previously . Briefly, animals were first allowed to acclimate to the testing environment (habituation). The test included two stages: training and testing. In the first stage of the test, the animal was confronted with two identical objects, placed in an open field, and in the second stage, the animal was exposed to two dissimilar objects placed in the same open field: one familiar object, used in the first phase, and the other novel object. Exploration of an object was defined as time spent with the head oriented towards and within two cm of the object. The time spent exploring each of the objects in stage two was detected by a video-camera using an EthoVision video tracking system (Noldus).
Morris water Maze
The classic Morris water maze (MWM) test was used to determine spatial learning and memory, as described previously [16, 19, 27–30]. Animals were initially randomized grouped to receive different treatments. The test was performed 24 hrs after last cessation of JZL184 injections. A circular water tank (diameter 120 cm) was filled with water and the water was made opaque with non-toxic white paint. A round platform (diameter 15 cm) was hidden 1 cm beneath the surface of the water at the center of a given quadrant of the water tank. PS19 TG mice and their age-matched littermates received learning acquisition training for 7 days (7 sessions), and each session consisted of 4 trials. For each trial, the mouse was released from the wall of the tank and allowed to search, find, and stand on the platform for 10 seconds within the 60-second trial period. For each training session, the starting quadrant and sequence of the four quadrants from where the mouse was released into the water tank were randomly chosen so that it was different among the separate sessions for each animal and was different for individual animals. The mice in the water pool were recorded by a video-camera using an EthoVision video tracking system (Noldus). A probe test to assess memory retention was conducted 24 hours after the completion of the training acquisition. During the probe test, the platform was removed from the pool, and the task performances were recorded for 60 seconds.
Data are presented as mean ± S.E.M. Unless stated otherwise, one- or two-way-analysis of variance (ANOVA) followed by post-hoc tests were used for statistical comparison when appropriate. Differences were considered significant when P < 0.05.