Mice and treatment

Mice were housed 3–4 mice per cage in a pathogen free mouse facility with ad libitum access to food and water on a 12 h light/dark cycle. All experiments included approximately equal ratio of male and female mice. The number of mice used for each experiment was specified in the figure legend. All procedures were performed in accordance with NIH guidelines and approval of the Baylor College of Medicine Institutional Animal Care and Use Committee (IACUC).

In vivo LPS treatment was performed as previously described [29]. Briefly, PPARαKO (Jackson Laboratory; Strain #:008154) and WT mice received a pretreatment of KDS-5104 (10 mg/kg, i.p.) or vehicle. 24 h post KDS-5104 treatment, mice received a co-treatment of KDS-5104 (10 mg/kg, i.p.) and LPS (2 mg/kg, i.p.). 18 h after the LPS injection, tissue was collected and fixed or frozen for further analysis.

For efficacy studies, two-month-old 5xFAD and WT littermate were treated with KDS-5104 (10 mg/kg, i.p.) or vehicle for three days a week for a total of 8 weeks. At the end of the treatment, behavioral assays were performed, and mice were sacrificed. The brains were perfused with PBS, dissected, and frozen or fixed for qPCR and biochemical analysis and immunostaining respectively.

In vitro cultures

The cellular model systems used include primary microglia cultures, BV2 cells and HeLa stable cell line containing the knockout of the three TFEB family genes; TFEB, TFE3 and MITF (TKO) [30].

Primary microglia monocultures were prepared from mixed gender PPARαKO and WT pups as previously described [31, 32]. Briefly, cortices were isolated from PPARα and wild type newborn pups (P0-P1) and cut finely in dissection media [Hank’s balanced salt solution (HBSS), 10% mM HEPES, and 1% (v/v) penicillin/streptomycin]. Next, tissue was digested with 2.5% trypsin for 15 min at 37oC before the addition of trypsin inhibitor (1 mg/ml) for 1 min. Tissue was then centrifuged for 5 min at 1500 rpm. Next, the pellet was triturated, resuspended in complete media (DMEM with 10% fetal bovine serum and 1% (v/v) penicillin/ streptomycin) and plated onto poly-d-lysine (PDL)–coated T-75 flasks at 50,000 cells/cm2 to produce mixed glial cultures. For microglia monocultures, mixed glia cultures were allowed to grow for one week, then microglia were separated from the mixed glia by shaking the confluent flask at 250 RPM for 2.5 h twice, 48 h between the two sessions. Following the final shaking, the flasks were tapped on a table and the floated cells, primarily microglia, were collected and seeded in PDL coated 12-well plate or glass coverslips at a concentration of 50,000 cells/cm2. Experiments were performed 24–48 h post seeding.

For KDS-5104 treatment, cells were plated 24 h prior to treatment. KDS-5104 was added to the complete media at a concentration of 10 µM unless otherwise stated. Eight hours after addition of KDS-5104, the cells were fixed or collected for further analysis. For PPARα antagonist experiments, cells were pretreated with 10 µM of GW6471 (Cayman Chemical) for 3 h prior to KDS-5104 treatment.

For LPS treatment, primary microglia or BV2 cell cultures were plated in 24 well plates with PDL-coated glass cover slips. Cells were treated with 10 µM of KDS-5104 or vehicle. Following an 8-hour incubation, media was replaced by LPS (5 µg/ml) and KDS-5104 (10 µM) containing media. Eighteen hours following LPS application, cells were fixed in 4% PFA in preparation for immunostaining procedures. For lipid droplet assay, following fixation cells were stained with BODIPY (Invitrogen) and washed with PBS three times. Coverslips were mounted and imaged using a Leica STELLARIS confocal microscope.

FACS based isolation of microglia from adult mouse brain

FACS sorting of microglia was performed as previously described with minor modifications [33]. Briefly, 9-month-old mice were perfused with PBS, brains extracted and gently minced with sterile razor blades. The tissue was digested in papain (Worthington Biochemical) and DNase (Worthington Biochemical), then titrated 5–6 times by a sterile fire-polished glass Pasteur pipette. Next, ice-cold HBSS+ (HBSS with 2mM EDTA and 0.5% BSA) was added and the suspension was pelleted at 310 g for 5 min at 4 °C. The pellet was resuspended in 1ml of HBSS+, triturated 5–6 additional times, and centrifuged. After centrifugation, the supernatant was filtered through a 40 μm cell strainer (BD Biosciences) and further centrifuged at 310 g for 5 min at 4 °C. The resulting pellet was resuspended in 20% 4 °C Percoll PLUS (Millipore-Sigma) in 1× PBS and centrifuged at 310 g at 4 °C for 20 min. The resulting pellet was incubated in 500ul HBSS + containing 1:100 Mouse BD Fc Block (BD Biosciences). Then with the following antibodies: rat anti–CD45-BV421 (1:500, BD Biosciences), rat anti–CD11b-FITC (1:500, BD Biosciences). Microglia population was gated and sorted based on CD45mid and CD11b+ expression. Sorting was performed using BD Biosciences Aria II on the 100 μm nozzle. Cells were sorted into 1.7 ml Eppendorf tubes coated with 200 µl HBSS+, followed by lysis of pellets in Qiagen RLT buffer containing 1% β-mercaptoethanol for downstream RNA analysis.

Phagocytosis assay

Phagocytosis assays were performed in primary microglia cultures as previously described [34]. Fluorescent latex beads were preprepared in FBS for 1 h at 37oC at a 1:5 ratio. The beads/FBS mixture was then added to prewarmed complete media (1:1000). Beads containing media were added to cells for 1 h, then removed and washed thoroughly with PBS. Cells were then fixed in 4% PFA and prepared for immunostaining. Cells were analyzed for percentage of microglia with beads internalization. For Aβ uptake and degradation, microglia were pretreated with CD36 neutralizing antibody (2 µg/ml; Abcam) as previously described [35]. Following blocking of CD36, microglia were treated with KDS-5104 for 8 h (10 µM). Meanwhile, fluorescently labelled Aβ42 (Cayman Chemical) was incubated in complete media for 37oC for 1 h at a concentration of 500 nM. Treated microglia were then incubated in Aβ containing media for 1 h at 37oC. Media were removed and cells washed. A portion of the cells were fixed at time point 0 and prepared for imaging. The remaining cells were fed with Aβ free fresh complete media for 1, 2 and 4 h. The cells were fixed, stained, and imaged on confocal with a 40x objective and the fluorescence of Aβ per cell was analyzed using the ImageJ software (NIH).

Western blotting

Brain tissue or cell pellets were lysed and homogenized in RIPA buffer containing protease and phosphatase inhibitors. Homogenates were centrifuged at 10,000 g for 15 min at 4oC and supernatant was collected. Bicinchoninic acid analysis (Thermo Fisher Scientific) was used to determine and normalize protein concentrations. Protein separation was performed by electrophoresis using 10–15% SDS-polyacrylamide gels. Following separation, proteins were transferred to a nitrocellulose membrane. Nonspecific binding was blocked by 5% BSA in tris-buffered saline then primary antibodies were incubated overnight at 4oC. Primary antibodies were used at the following concentrations; PPARα (1:1000; Cell Signaling), APP (1:1000; recognizes APP-FL and CTF, Cell Signaling), β-actin (1:10,000; Sigma-Aldrich), pS6k (1:1000; Cell Signaling), total S6k (1:1000, Cell Signaling), pAKT (1:1000, Cell Signaling), total AKT (1:1000, Cell Signaling) and γ tubulin (1:1000, Cell Signaling). After primary antibody incubations, secondary antibodies; IR-680–conjugated goat anti-mouse or goat anti-rabbit (1:10,000; Molecular Probes) and IRDye 800–conjugated donkey anti-rabbit or donkey anti-mouse (1:10,000; LI-COR) were used. LI-COR Odyssey machine (LI-COR) was used to image the membranes. The Western blot bands were quantified using the ImageJ software.

RNA isolation, reverse transcription and qPCR

Total RNA was isolated from cells, human and mouse brain tissues by lysing in Qiagen RLT buffer with 1% β-mercaptoethanol and processed using the RNeasy Mini kit (Qiagen). Reverse transcription was carried out on the isolated RNA using iScript Reverse Transcription Supermix (Bio-Rad). The qPCR analyses were performed using iTaq Universal SYBR Green master mix (Bio-Rad) on a CFX384 Touch Real-Time PCR Detection System. Housekeeping genes 18s and GAPDH were used as controls. Relative levels of gene expression were quantified by the Bio-Rad CFX manager. Heatmaps were constructed using GraphPad Prism.

Immunofluorescence

Cells were fixed in 4% PFA and prepared for immunocytochemistry. Briefly, blocking buffer (0.2% BSA, 0.5% Triton X-100, and 0.05% Tween 20 in PBS) was used to block nonspecific binding sites for 1 h at room temperature. Next, cells were incubated with primary antibodies: Iba-1 (1:800 or 1:500; Wako or Novus Biologicals, respectively), CD36 (1:1000; Abcam), LAMP1 (1:500; BD Biosciences). Following primary antibody incubation, cells were incubated with appropriate secondary antibodies (Alexa Fluor 488, 555, or 647; Invitrogen). The nucleus was then stained with 4′,6-diamidino-2-phenylindole (DAPI). Glass cover slips were then mounted and imaged under the confocal microscope. For lysotracker staining, prior to fixation, cells were incubated in complete media containing lysotracker dye (50 nM; Thermo Fisher Scientific) for 30 min at 37oC. Cells were thoroughly washed in PBS and fixed for 15 min in 4% PFA. Coverslips were mounted and imaged.

For mouse brain tissue, immunohistochemistry was performed on free floating brain sections. Briefly, mice were perfused with saline, brains quickly extracted and fixed in 4% PFA at 4oC overnight. Brains were then transferred to 30% sucrose for 48 h and microtome-cut into 30 μm thick sections. Free floating sections were incubated with primary antibodies: 6E10 (1:1000; BioLegend), Iba1 (1:800 or 1:500; Wako or Novus Biologicals, respectively), GFAP (1:1000; Sigma), CD68 (1:500; BioLegend), PSD95 (1:200; Millipore), synaptophysin (1:500; Abcam). Next, appropriate secondary antibodies (Alexa Fluor 488, 594, or 647; Invitrogen) were used followed by incubation with DAPI. Three sections (including hippocampus and cortex) per mouse brain with five to seven mice per group were used. Brain sections from treatment groups were coded prior to imaging to reduce imaging bias.

Image quantification Synaptic co-localization

Synaptic marker co-localization analysis was performed with the Imaris software (Oxford Instruments) as described previously [29, 32]. Briefly, synaptophysin (Abcam) and PSD95 (Millipore), respective markers for pre- and post-synaptic terminals, were stained in mouse brain sections as described above. Sections were imaged with a 63X oil objective with a 4.0 digital zoom on the Leica STELLARIS confocal microscope. 5 μm thick Z stacks with 0.2 μm step size were obtained. Using the Imaris ‘Spots’ feature, puncta from each channel were analyzed by generation of spot representation (automatic generation with consistent manual adjustment for all images for accuracy). Total number of spots for each channel were recorded. Spots were then analyzed using the ‘Co-localize Spots’ MATLAB plugin, defining co-localization if the center of the pre- and post-synaptic puncta were within 200 nm.

Lysosome characterization

For analysis of LAMP1 and lysotracker images (obtained with 63x objective, 2x digital zoom) the Cells feature of Imaris was used. Cell borders and nucleus were defined followed by the generation of representative spots for each marked lysosome (automatic generation with consistent manual adjustment for all images for accuracy). Spots were then analyzed for size, fluorescent intensity, proximity to nucleus and number per cell.

Lipidomics

Brain tissue from 4-month-old WT-vehicle, WT-KDS-5104, 5xFAD-vehicle and 5xFAD-KDS-5104 mice were weighed and normalized. Tissue was then ground using a bead beater. 10 mg of each sample was collected and homogenized in 50 mM ammonium acetate solution. 10 µl of splash lipidomix Mass Spec Standard (Avanti, 330,707) was added to each sample. Following standard addition, lipids were extracted using methanol, metyl terrt-Butyl Ether (MTBE) and water. The samples were then dried in a vacufuge and resuspended in 110 µl isopropanol and methanol (50:50, vol/vol). The samples were analyzed using a Vanquish UPLC and a Lumos orbitrap mass spectrometer (Thermo Fisher Scientific). Analysis of lipidomic data were performed using Lipidsearch software (Thermo Fisher Scientific). The statistical analysis was done by MetaboAnalyst 5.0.

Behavioral assays

At the conclusion of animal treatments with vehicle or KDS-5104, mice underwent behavioral testing. Mice treatment groups were coded and blinded to researcher performing and analyzing results. Prior to each assay, mice were habituated to the test room for 30 min.

Novel object recognition: The novel object recognition protocol included three phases: habituation, a training, and object recognition. All three phase are performed in a Plexiglass arene (measuring 22 cm by 44 cm). The habitation phase included one 5-minute session, in which the animals were allowed to freely explore the arena. Twenty-four hours post habituation, the animals underwent training, in which the mice were placed in the arena with two identical objects. The animals were allowed to freely explore the objects for 5 min. One day after the training phase, the mice underwent testing, in which the mice were placed in the same arena with one object previously explored in the training phase, the familiar object, and one novel object differing in color and shape but sharing a common size and volume. The animals were allowed to freely explore the objects for 5 min. The time spend exploring each object was measured by the ANY-maze software. Exploration of an object was defined by head orientation directed toward the object or physical contact with the object. The object discrimination ratio (ODR) was calculated by the following formula: ODR = (time exploring specified object) / (time exploring novel object + time exploring familiar object) × 100.

Fear conditioning

The fear conditioning protocol involved a training phase, context test, and a cued test. During the training phase, the mice were placed in the conditioning chamber and allowed to freely explore. At 3 min, an auditory stimulus was presented for 30 min (80-dB white noise) followed by the administration of a foot shock (0.8 mA, 2 s). This was repeated a second time at the 5-min mark. Following training, the mice were then returned to their original housing cages for 24 h before performing context and cue testing. For the context test, each mouse was returned to the same chamber (same geometric shape of chamber, lights, scents, and auditory sounds) for 5 min with no stimulation, freezing was recorded. The cue test was then performed, one hour after the context test. For the cue test, mice were placed in an altered chamber consisting of a different geometric shape, flooring, light brightness, and scent compared to the previous chamber used for training. After 3 min, the auditory stimulus was presented for 3 min. The software FreezeFrame3 and FreezeView (San Diego Instruments) was used to record and analyze the percent freezing in each trial. Methods for open field, grip strength and rotarod tests are described in Supplementary Materials.

Statistical analysis

All data were analyzed with GraphPad Prism version 6 and presented as means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). For simple comparisons, Student’s t-tests was used. For multiple comparisons, analysis of variance (ANOVA) followed by Tukey’s multiple comparisons tests as the post hoc analysis were performed. To address sex as a biological variable in the 5xFAD mouse model secondary analysis by two-way ANOVA with sex as a between-subjects factor and treatment group as a within-subjects factor followed by post hoc analysis. All samples or animals were included in the statistical analysis unless otherwise specified.