The NLRP3 inflammasome inhibitor OLT1177 rescues cognitive impairment in a mouse model of Alzheimer’s disease

Significance IL-1β is an immunomodulatory cytokine that is overexpressed in the brains of patients with Alzheimer’s disease (AD). The NLRP3 inflammasome is an intracellular complex that activates caspase-1, which processes the IL-1β and IL-18 precursors into active molecules. In this study, we used an APP/PS1 mouse model for AD, which confirms significant cognitive losses that are recovered in NLRP3-deficient mice, to evaluate the therapeutic potential of an orally bioavailable and safe NLRP3 inhibitor, OLT1177. OLT1177 ameliorated the phenotype in APP/PS1 mice, as evidenced by rescued spatial learning and memory in the Morris water maze test. Microglia were less activated, cortical plaques reduced, and plasma AD metabolic markers were normalized. OLT1177 is a potential therapeutic option for AD.

Morris Water Maze test. Spatial memory formation and retention was assessed using the Morris water maze (MWM) assay (1). In this test, a 10 cm escape platform was submerged 1 cm below the water surface into a circular plastic pool (160 cm in diameter and 60 deep) filled up to 30 cm with opaque water (titanium dioxide, Euro OTC Pharma; water temperature 19-20 °C). Three visual cues were positioned on the walls around the pool. All tests were performed under dim light at the same time of day during the light period between 9:00 to 16:00 by an experimenter blind to all groups. A digital camera was installed above the center of the maze and videos were acquired and transmitted to a PC running the tracking software ANY-maze (Stoelting, USA). Prior to the training, a visible platform task was performed as a pre-training and used to ensure that swimming ability and visual acuity were intact in all experimental groups, moreover, this phase was important for the animals to get accustomed to the test situation. During this phase, the animals had two trials (maximum of 60 s each) per day for three consecutive days to reach the visible platform, the position was alternated during the trials as is standard practice.
Subsequently, training in the Morris water maze was performed for 8 days with the invisible platform located in the northwest (NW) quadrant. Each day, animals were placed in the water for four trials, with different starting points (SW, S, E, and NE) randomly at a 5 min intertrial interval. The animals were permitted to swim freely for 60 s or until the platform was reached. Otherwise, they were guided to the platform and allowed to sit on it for 15 s. For evaluation of reference memory, two probe trial tests were performed at the third day of the acquisition training, prior to the training session on that day. Another reference memory test was performed 24 h after the last day of acquisition training (day 9). During the probe trial, the platform was removed and the animals were allowed to swim freely for 45 s (starting position SE).

Electrophysiological experiments.
To study the function of CA1 hippocampal neurons, electrophysiological recording experiments were caried out in different experimental groups. For this purpose, acute hippocampal slices were prepared from mice in four groups including wild-type, APP/PS1, APP/PS1 treated with 3.75 g and APP/PS1 treated with 7.5 g of OLT1177. Briefly, mice were deeply anesthetized with 100% CO2, killed, and then brains were quickly removed and transferred into ice-cold carbogenated (95% O2 and 5% CO2) artificial CSF (ACSF) containing the following (in mM): 124 NaCl, 4.9 KCl, 1.2 KH2PO4, 2.0 MgSO4, 2.0 CaCl2, 24.6 NaHCO3, and 10 D-glucose, pH 7.4. Afterward, the hippocampus was dissected and transverse hippocampal slices (400 μm) were obtained using a manual tissue chopper. The whole procedure was done in 2-3 min. The hippocampal slices were transported to an interface recording chamber (Scientific System Design) and they were incubated at 32°C with a constant flow rate (0.5 ml/min) of carbogenated ACSF for 2 h prior to the start of recordings. Field excitatory post synaptic potentials (fEPSPs) were recorded in the stratum radiatum of the hippocampal CA1 sub-region. Responses were evoked by stimulation of the Schaffer collateral pathway using two monopolar, lacquer-coated stainless-steel electrodes (5 MΩ; AM Systems). These stimulation electrodes (S1 and S2) were placed equidistantly on both sides of the recording electrode and, by this means, two independent stimulation pathways could be used for the same CA1 recording region. For recording fEPSPs (measured as the first slope function), the recording electrode (5 MΩ; AM Systems) was positioned in the CA1 apical dendritic layer (at least 20 μm away from the stratum pyramidale) and signals were amplified by a differential amplifier (Model 1700; AM Systems). The signals were digitized using a CED 1401 analog-to-digital converter (Cambridge Electronic Design). An input-output curve (afferent stimulation vs fEPSP slope) for assessment of basal synaptic transmission was generated after the pre-incubation period. Test stimulation intensity was modified to be adjusted to extract fEPSP slope as 40% of the maximal fEPSP response for both synaptic inputs S1 and S2. To investigate long-term potentiation, 20 min after baseline recording, LTP was induced by theta-burst stimulation (TBS) including four bursts at 100 Hz repeated 10 times in a 200 ms interval. This stimulation was repeated three times in a 10 s interval. Only healthy sections with a stable baseline were included in the electrophysiological data analysis. The slope of fEPSPs was measured over time for 60 min and normalized to the baseline. Data acquisition and offline analysis were caried out using IntraCell software (version 1.5, LIN) (2, 3).
Morphological analysis of hippocampal neurons: Golgi-Cox staining. For morphological quantification of hippocampal neurons, Golgi-Cox staining was performed. For this purpose, mice were deeply anesthetized with CO2 and sacrificed. The left hemisphere was incubated in FD rapid Golgi-Cox stain kit according to the manufacturer's protocol. Briefly, each hemisphere was immersed in 2 ml impregnation Golgi solution, a mixture of solutions A (potassium dichromate and mercuric chloride) and B (potassium chromate) for at least 14 days to 1 month at room temperature in the dark followed by one week in tissue-protectant solution C at room temperature. Both impregnation and solution C were replaced with fresh solutions on the second day. Subsequently, hemispheres were blocked in 2% agar and using a vibratome (Leica VT 1000 S) 200 μm thick coronal sections were cut. The slices were collected on gelatin-coated glass slides. Afterward, the sections were processed with solutions D and E for signal development for 10 minutes before being dehydrated through graded alcohols and mounted using Permount (Thermo Fisher Scientific). The slides were then kept in a horizontal position for drying in the dark for at least 48 h before imaging (2, 3).
Imaging and image analysis. To survey the hippocampal neuron morphology, second-or third-order branches of both apical and basal dendrites within the pyramidal shaped CA1 neurons (8 cells in each subregion per animal) were imaged in three-dimensions (z-stack thickness of 0.5 μm) using an Axioplan 2 imaging microscope (Zeiss) equipped with a 63x (N.A. 1) oil objective accompanied with a digital camera (AxioCam MRm, Zeiss). In all selected neurons, spine density per micrometer of dendrites was calculated using Fiji software (BioVoxxel) on the segments of dendritic branches with a length of more than 60-70 µm which were positioned at least 40-50 μm away from the cell soma. The total number of spines along the segment of dendritic branches was counted manually. All slides were coded and analyses were performed blindly (2, 3).
The microscopic images of anti-IBA-1 and amyloid-β (clone BAM-10) were taken within the area of cortex and hippocampus (3 slices per animal). To survey the hippocampal microglial morphology, images of IBA-1 cells were taken from the CA1 area of the hippocampus in 3D (z-stack thickness 1 µm) using Axioplan 2 imaging microscope (Zeiss) equipped with an ApoTome module (Zeiss) with a 20X objective (NA, 0.8) and a digital camera (AxioCam MRm; Zeiss). To analyze the density of microglia cells, a region of interest (ROI) was drawn in each frame of the hippocampus and the IBA-1 positive cells were determined with the clearly visible nuclei by DAPI and ImageJ software (Wayne Rasband NIH, USA). To investigate the activation status of microglia cells primary processes of IBA-1 positive cells were counted with ImageJ software.
Metabolomics analyses. Plasma (10 µl) metabolomes were characterized by ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry (Vanquish -Q Exactive, Thermo Fisher, San Jose, CA). Extraction was performed at a 1:10 ratio in ice cold methanol:acetonitrile:water 8:3:2 v/v via vortexing for 30 min at 4°C, as previously described (4). Analytical details of chromatographic gradients and instrument operation settings were identical to those described extensively in prior methodological and application papers (4,5) and are here omitted in the interest of space.
Statistical analysis. Data were analyzed and plotted by GraphPad Prism 6 (GraphPad Software, Inc. USA) and presented as mean±SEM. Differences in dendritic spine density, immunostaining and cytokines measurement data were subjected to a one-way ANOVA whereas two-way ANOVA was used for behavioral and electrophysiological experiments. Fisher's LSD, Bonferroni's and Tukey's multiple comparisons were used as a post hoc test depending on experiments. The minimum significance value was considered as p < 0.05. All statistical analysis and n of different experimental groups were reported in the results and figure legends respectively.    Bonferroni's multiple comparison: APP/PS1 vs APP/PS1 7.5 g/kg OLT1177 p = 0.011 H; n = number of sample WT n = 6, APP/PS1 CTRL n = 10, APP/PS1 3.75 g/kg n = 12, APP/PS1 7.5 g/kg n = 10). (I) The amount of Aβ plaques was determined using immunostaining for IBA-1 and BAM-10 (clone for amyloid-β) (IBA-1 in green, DAPI in blue, BAM-10 in red; scale bar 500 µm). (J) Plaque load was lower in the cortex of the APP/PS1 animals fed with 7.5 g/kg OLT1177 compared to APP/PS1 animals (Cortex Unpaired t-test: t = 2.476, df = 10, p = 0.03; Hippocampus: Unpaired t-test: t = 1.484, df = 10, p = 0.16, n = number of sample n = 6 in both groups). Data are presented as mean ± SEM. *** p < 0.001 compared to WT, +++ p < 0.001 compared to APP/PS1 CTRL.  significant metabolites by ANOVA revealed a significant effect of APP/PS1 on plasma metabolism compared to controls. (C-E) Significant effects of APP/PS1 and OLT1177 treatment were noted with respect to carboxylic acids (C), glutaminolysis (D), deaminated purines (e.g., allantoate), glutathione turnover metabolites (5-oxoproline), proteolysis (including the urea cycle intermediate ornithine), and tryptophan catabolism (kynurenine -E). (F) Polyunsaturated fatty acids were decreased in the bloodstream of APP/PS1 mice and normalized by 7.5 g/kg OLT1177. (G) An overview of the overall impact of APP/PS1 and OLT1177 (higher dose) on mouse plasma metabolism is provided in H. Data are presented as mean ± SEM. *** p < 0.001 compared to WT, +++ p < 0.001 (one-way ANOVA and with multiple column comparison) N = 3 animals.