Morris Water Maze Test

Caspase-1 activation of human and mouse brain tissue were analyzed by Western blot of cleaved caspase-1. IL-1β was quantified by ELISA. Microglial ASC speck formation was detected by immunohistochemistry. All mice were on C57/Bl6 background, including WT, NLRP3^{−/−,27 (link)}, APP/PS1^{5 (link)}, APP/PS1/NLRP3^−/−, Caspase-1^{−/−,28 (link)}, APP/PS1/Caspase-1^−/− and were analyzed for cognitive function using the Morris Water Maze, the object recognition test and open field behavioural testing. Synaptic plasticity was determined by measuring long term potentiation (LTP) in acutely isolated hippocampal slices. Spine density was assessed by analyzing mid apical dendritic sections of pyramidal CA1 neurons. Cerebral Aβ load was determined by thioflavin-S-histochemistry of serial sections. Sequential extraction of homogenized brains by radio-immunoprecipitation assay, sodium dodecyl sulfate buffer and formic acid was employed to determine Aβ levels. Aβ nitration was determined by ELISA and immunohistochemistry using a specific antibodies against 3NTyr^{10 (link)}-Aβ^{25 (link)}. Western blot detection was used to analyze the protein levels of APP, CTFs, Aβ, BACE1, IDE and NOS2. Inflammasome activation was confirmed by detection of ASC speck formation in microglia isolated from adult mouse. Microglial Aβ phagocytosis was determined after peripheral injection of methoxy-XO4, isolation of microglia and subsequent FACS analysis. Confirmatory immunocytochemistry was performed using antibody IC16 and the lysosomal marker LAMP2. Plaque morphology and microglial Aβ uptake was analyzed by coimmunostaining with Iba-1, methoxy-XO4 and IC16. mRNA levels of IDE, NEP, M1 and M2 markers were determined either from sorted microglia or from brain tissue by qPCR.

Five- to six-month-old male Thy1-hAPP^Lond/Swe+ mice and their wild-type littermates were used in this study. Transgenic lines were maintained by crossing heterozygous Thy1-hAPP^Lond/Swe+ mice with C57BL/6J breeders. Littermate cage-mates were used as control mice. Five cohorts of mice with an n between nine and 19 were used. The total number of mice used was 68 controls and 65 mutants. The genotype of all animals was determined by PCR before experiments. All transgenic mice were heterozygous with respect to the hAPP^Lond/Swe gene. Twelve-week-old male C57BL/6J mice (from Jackson Laboratory, Bar Harbor, ME) were used for the validation of the delayed-matching-to-place (DMP) dry maze task. All animals were housed in a 12-h dark/light cycle, temperature- and humidity-controlled environment with unlimited access to water and food. The same group of animals was tested in the activity chamber, open field, and fear conditioning (FC). Different groups of mice were used for the social interaction tests, Morris water maze (MWM), DMP water maze, DMP dry maze, and hot plate test. Experimenters were blind to the genotype of the mice throughout testing. All tests were conducted in the light cycle. In all experiments, animals were habituated to the testing room 2 h before the tests and were handled by the experimenter for five days before all the behavioral tests. All experiments were in accordance with protocols approved by the Institutional Animal Care and Use Committee of Stanford University and were performed based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All actions were considered for reducing discomfort of animals during all experiments.