Chemicals

Acetonitrile, methanol, isopropanol, water, methyl-tert butyl ether and ammonium acetate (LC-MS grade) were purchased from Sigma Aldrich (St. Louis, MO, USA), internal standard mixture SPLASH® LIPIDOMIX® was obtained from Avanti Polar Lipids (Alabaster, Alabama, USA) and internal standards: Creatine-d3 (methyl-d3) from CDN isotopes (Pointe-Claire, QC, Canada), L-Leucine-5,5,5-d3 and Butyryl-L-carnitine-(N-methyl-d3) hydrochloride, both from Sigma Aldrich (St. Louis, MO, USA).

Animals

All experiments were performed on Tg SHR-24 rats expressing human truncated tau aa151-391/3R. As controls, we used age-matched non-Tg animals (SHR). Animals included in this study were 4, 6, 8, 10, 12, and 14 months old. All animals were bred in the in-house animal facility of the Institute of Neuroimmunology of the Slovak Academy of Sciences in Bratislava. All animals were housed under standard laboratory conditions with free access to water and food and were kept under diurnal lighting conditions (12 h light/dark cycles with light starting at 7 am). All experiments were performed according to the institutional animal care guidelines and following international standards (Animal Research: Reporting of In Vivo Experiments guidelines) and approved by the State Veterinary and Food Administration of the Slovak Republic (Ro-933/19–221/3a) and by the Ethics Committee of the Institute of Neuroimmunology, Slovak Academy of Sciences. Efforts were made to minimize the number of animals utilized and limit discomfort, pain, or any other suffering of the experimental animals in this study.

Human tissue specimens

Human brain tissue samples were obtained from the Queen Square Brain Bank for Neurological disorders (London, UK). All samples were obtained with informed patient consent and approval from the local ethical committees.

Collection of samples

For the biochemical, metabolomics, and lipidomics experiments, animals were deeply anesthetized with tiletamine-zolazepam/xylazine anesthesia (4/2 mg/kg). From every rat, we collected plasma, CSF, and brain tissue. CSF samples were collected from cisterna magna in an amount ranging from 80 to 120 µL. Anesthetized animals were fixed in a head holder, and a midline incision in the skin was made up to the head area to permit easy access to the cisterna magna. After the centrifugation (4,000xg, 10 min, 4 °C) samples were immediately flash-frozen in liquid nitrogen and stored at -80 °C until further use. Approximately 4 ml of blood was collected from the heart to vacuum blood collection tubes with EDTA and centrifuged (4,000xg, 10 min, 4 °C). Collected plasma samples were immediately frozen in liquid nitrogen and stored at -80 °C. Brain tissue was cut sagittally and divided into different anatomical regions – pons and medulla oblongata. Every brain region, the left and right hemispheres, was transferred into separate tubes, flash-frozen immediately in liquid nitrogen, and stored at -80 °C.

Sample preparation

Firstly, each tissue was homogenized using homogenizer FastPrep-24 in 300 µL of methanol spiked with an internal standard mixture SPLASH® LIPIDOMIX® (2.5%). After that, 1 ml of methyl-tert butyl ether was added, and the sample was vortexed for 10s. Then, 300 µL of water was added to form a two-phase system, vortexed, and left vibrating for 15 min at room temperature (RT). The samples were subsequently left to equilibrate for 15 min at 4 °C then centrifuged at 10,000xg for 10 min at 4 °C. At last, 400 µL of upper phase was collected for lipid analysis, and a mixture of upper and lower phase (1:1 v/v) for analysis of metabolites. Collected extracts were left to freeze at -80 °C for 20 min and freeze-dried overnight. Freeze-dried samples for lipid analysis were reconstituted in 100 µL of acetonitrile/isopropanol/water (2:2:1 v/v/v), centrifuged at 10,000xg for 10 min at RT and 80 µL of supernatant was collected and transferred into LC-MS grade glass vials. For the preparation of quality control (QC) samples, 5 µL of each supernatant was mixed to form a pooled sample. Freeze-dried samples for metabolomic analysis were reconstituted in a 100 µL mixture of water/methanol (1:4 v/v). The methanolic part of the mixture contained isotopically labeled internal standards: leucine (2.0 µM), butyryl carnitine (0.2 µM), and creatinine (2.0 µM). After centrifugation (15,000xg, 15 min, 4 °C) supernatants were transferred into vials, and a QC sample was prepared the same as in the sample preparation procedure for lipid analysis. CSF and plasma samples for lipid analysis were prepared by adding 80 µL of isopropanol and 2 µL of internal standard mixture SPLASH® LIPIDOMIX® (2.5%) to 20 µL of each sample, vortexed and kept overnight at -80 °C for deproteinization. After centrifugation (15,000xg, 10 min, RT) 80 µL of supernatant was collected and transferred into a glass vial which was used for the subsequent LC-MS analysis. For analysis of metabolites, CSF and plasma samples were prepared by adding 20 µL of sample to 80 µL of the methanolic solution containing selected labeled internal standards, vortexed, and kept overnight at -80 °C for deproteinization. Subsequently, mixtures were vortexed and centrifuged (15,000xg, 5 min, 4 °C). The supernatant was transferred into vials and analyzed. The pooled QC samples for each sample type were prepared by collecting 5 µL of each sample supernatant and pooled into one.

LC-MS analysis

Targeted lipidomic analysis was carried out by a pseudotargeted approach adopted from [17] using a liquid chromatography-tandem mass spectrometry system consisting of ExionLC™ for UHPLC and QTRAP® 6500 + for MS/MS (Sciex, USA). A reversed-phase ACQUITY BEH C8 column (2.1 × 100 mm, 1.7 μm, Waters) was used for chromatographic separation. Mobile phase A consisted of acetonitrile/water (60:40 v/v), and mobile phase B consisted of isopropanol/acetonitrile (90:10 v/v) both containing 10 mM ammonium acetate. The column temperature was maintained at 55 °C and the sample manager temperature was set at 15 °C. Gradient starting conditions were 32%B (0–1.5 min) with a gradual increase to 85%B (1.5–15.5 min), and a further steep rise to 97%B (15.5–15.6 min). 97%B was maintained until 18.0 min followed by a re-equilibration step at 32%B (18–20 min). The total run time was 20 min at a flow rate of 0.35 mL/min. Mass spectra were acquired simultaneously in both positive and negative modes in the scheduled multiple-reaction monitoring (scheduled MRM) mode. The settings for the mass spectrometer were as follows: capillary voltages were set to + 5500 V/-4500 V, the pressure of curtain gas to 40 psi, and the source temperature was 500 °C. Samples were measured in randomized order with continuous monitoring of QC samples. Confirmation of correct identification of lipids was evaluated using lipid elution pattern plots, generated by R script and the same workflow as in Drotleff, 2020 [18]. Targeted metabolomic analysis adopted from Yuan, 2012 [19] and performed using the same LC-MS instrumentation as in the lipidomic analysis. Identification of metabolites and optimization of their measurement parameters were based on standard compounds as was already described in our previous work [20]. The aminopropyl column (Luna 3 μm NH2, 2 × 100 mm, Phenomenex) was used for the chromatographic separation. Mobile phase A consisted of 20 mM ammonium acetate (pH 9.75), and acetonitrile was used as mobile phase B. The column temperature was maintained at 35 °C. Gradient starting conditions were as follows: 95% − 10%B (0–7 min), 10% B (7–13 min), 95%B (13–17 min). The total analysis time was 17 min with a flow rate of 0.3 mL/min. Mass spectra were acquired in both positive and negative modes in the scheduled MRM mode. The settings for the mass spectrometer were as follows: capillary voltages were set to + 5500 V/-4500 V, the pressure of curtain gas to 40 psi, and the source temperature was 400 °C. The data were acquired using Analyst software (v1.6.2) and processed using MultiQuant v3.0 (both Sciex, USA) for both metabolomics and lipidomics. All samples were measured in randomized order with continuous monitoring of QC samples.

Neurofilament-light-chain and tau quantification

Concentrations of neurofilament light chain (NFL) in plasma and tau proteins (total-tau) in CSF and cell culture media were measured using single-molecule array (Simoa) digital ELISA, using an HD-1 Analyzer. For the analysis, Simoa™ NF-Light Advantage Kit, and Simoa™ Mouse TAU Discovery Kit were used. Briefly, frozen blinded samples were melted, centrifuged for 10 min at 25 °C for 20,000xg, and supernatants were transferred into a Simoa sample plate together with calibrators for NFL and Mouse TAU kits (parts of kits). Each measurement was done in duplicate according to the manufacturer’s recommendations. Concentrations were calculated using Simoa™ HD-1 instrument software. The concentration of human tau released into cell culture media (proline-rich domain) was measured by in-house developed ELISA assay using the DC116 (aa 188–224) and DC242 (aa156-180) antibodies (Axon Neuroscience R&D Services SE, Bratislava, Slovakia).

Cultivation and LPS stimulation of BV2 cells

Mouse microglial BV2 cells (C57BL/6, purchased from ICLC, Modena, Italy) were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM, PAA Laboratories GmbH, Colbe, Germany) containing 10% fetal calf serum (FCS, Invitrogen, Carlsbad, California, USA), and 2 mM L-glutamine (PAA laboratories GmbH, Colbe, Germany) at 37 °C and 5% CO2. The medium was changed twice a week. One day before treating the BV2 cells with recombinant tau protein (aa 151–391/3R), the cell culture medium was replaced with a serum-free DMEM medium with L-glutamine. To stimulate the cells, tau protein was added for a final molarity of 500 nM for 24 h.

Isolation and cultivation of primary rat glial culture

Rat mixed glial culture was prepared from cerebral cortices of 1–2 day old Sprague Dawley rats (n = 8/isolation). The animals were euthanized by CO2, and the cerebral cortices were dissected, stripped of the meninges, and mechanically dissociated by repeated pipetting followed by passage through a 20 μm nylon mesh (BD Falcon, Franklin Lakes, USA). Cells were plated on 6-well plates pre-coated with poly-L-lysine (10 µg/mL, Sigma-Aldrich, St. Louis, MO) and cultivated in DMEM medium containing 10% FCS and 2 mM L-glutamine at 37 °C, 5% CO2 in a water-saturated atmosphere.

Development of a multi-component cell model system

The multi-component model was composed of rat primary microglia, astroglial cells, and human neuroblastoma cell line SH-SY5Y with inducible expression of truncated tau protein (previously described by [21]). Firstly, the expression of truncated tau protein was induced by cultivating cells in a medium without doxycycline (Sigma-Aldrich, St. Louis, MO) for three days before cell seeding into co-culture. Secondly, SH-SY5Y cells were co-cultivated with primary glial culture in 6-well plates in density 1.103 cells/cm2 for a further 5 days in DMEM medium containing 10% FCS, 2 mM L-glutamine and N2-supplement (Life Technologies Invitrogen, Carlsbad, CA) at 37 °C and 5% CO2. The medium was changed twice a week. 24 h before the experiment, we cultivated the cells in a medium with 2% fatty acid-free BSA (Life Technologies Invitrogen, Carlsbad, CA).

Isolation of lipid droplet-associated mitochondria (LDM)

The brainstem from Tg and control rats (n = 4) was harvested and washed with phosphate buffer saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) to remove blood contamination. The brain tissue was suspended in sucrose-HEPES-EGTA buffer supplemented with 2% BSA (250 mM sucrose, 5 mM HEPES, 2 mM EGTA, 2% fatty acid-free BSA, pH 7.2). The tissue suspension was homogenized and the homogenate was transferred into a 15 mL falcon tube. Homogenate was centrifuged in a swinging bucket rotor at 900xg for 10 min at 4 °C. The post-nuclear supernatant was collected and layered with Buffer B (20 mM HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4). The second centrifugation was carried out in a swinging bucket rotor at 2,000xg for 40 min at 4 °C to allow the fat layer to separate. The layer that contains LDs appears as a translucent band on the top of the gradient. This layer was resuspended in HEPES buffer without BSA and centrifuged at 10,400xg for 10 min at 4 °C in a fixed rotor. The LDM pellet was resuspended in mitochondrial resuspension buffer (250 mM mannitol, 5 mM HEPES, 0.5 mM EGTA, pH 7.4).

ATP assay

The assay was performed using a Luminescent ATP detection assay kit from Promega (Wisconsin, USA). 50 µL of isolated lipid droplet mitochondria (LDM) were suspended in 50 µL of 20 mM Tris pH 7.5 buffer and combined with 100 µL of CellTiter-Glo® 2.0 Reagent in an ELISA standard plate. The plate was placed on an orbital shaker for 5 min, and luminescence was recorded immediately thereafter using Luminometer, Fluoroscan Ascent FL (ThermoScientific, US).

AlamarBlue assay

AlamarBlue™ solution was added to the cell culture in dilution 1/10. Cells were incubated at 37 °C and 5% CO2 for 4 h. 100 µl aliquots of each sample were transferred to replicate wells of a black 96-well plate. The fluorescence was measured at 590 nm, using an excitation wavelength of 530–560 nm.

Immunocytochemistry and BODIPY staining

BV2 and primary rat glial cells were fixed for 15 min in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and washed with PBS. Cells were blocked for 60 min in 5% BSA in PBS. Cells were washed with PBS and incubated with primary antibody rabbit polyclonal anti-IBA-1 (FUJIFILM Wako Chemicals, USA) overnight. After washing, the cells were stained with BODIPY 493/503 (1:1000, Thermo Fisher) for 15 min at RT. Cells were mounted and examined using an LSM 710 confocal microscope (Zeiss, Jena, Germany).

Immunohistochemistry

Animals were anesthetized by tiletamine-zolazepam/xylazine anesthesia and perfused intracardially with PBS with heparin. The brain was removed and embedded in a cryostat embedding medium (Leica, Wetzlar, Germany) and frozen above the surface of liquid nitrogen. 12-µm-thick brain sections were cut on a cryomicrotome, fixed onto poly-L-lysine coated slides, and left to dry at RT for 1 h. Sections were fixed for 15 min in 4% paraformaldehyde and blocked for 60 min in a blocking solution (DAKO, Ontario, Canada). Sections were incubated overnight in a polyclonal rabbit anti-IBA-1 primary antibody (1:1000, Wako), polyclonal rabbit anti-GLUT3 primary antibody (1:100, Invitrogen, Massachusetts, USA), and monoclonal mouse anti-NeuN primary antibody (1:1000, Abcam, Waltham, MA, USA). After washing, the sections were incubated in goat anti-rabbit or anti/mouse AlexaFluor546/488 secondary antibodies (1:2000, Invitrogen, Massachusetts, USA) for 1 h at RT. Sections were washed with PBS and incubated with BODIPY 493/503 in PBS (1:1000, Invitrogen, Massachusetts, USA) for 15 min at RT. For tau staining, sections were incubated in monoclonal mouse anti-rat pSer202/pThr205 primary antibody (1:1000, Invitrogen, Massachusetts, USA). After washing, the sections were incubated for 1 h in a secondary antibody goat anti-mouse AlexaFluor488 (1:1000, Invitrogen, Massachusetts, USA). Brain slices were mounted (Vector Laboratories, Burlingame, CA, USA) and examined using an LSM 710 confocal microscope. Oil Red O (Abcam, Waltham, MA, USA) was used for the staining of neutral lipids. The slides were incubated in propylene glycol for 5 min and then in heated Oil Red O solution for 10 min. The sections were differentiated in 85% propylene glycol for 1 min and washed twice in water. The brain slices were mounted in an aqueous mounting medium.

Measurement of membrane fluidity

Membrane fluidity was measured by lipophilic pyrene probe - pyrenedecanoic acid (PDA, Abcam, Waltham, MA, USA), which undergoes dimerization after the interaction and exhibits changes in its fluorescent properties. BV2 cells were treated with tau protein (final concentration 1 μm) for 24 h. The cells were incubated with PDA/Pluronic F127 solution for 1 h at 25 °C in the dark with gentle agitation. The unincorporated PDA was removed by washing cells twice with cultivation media. The live-cell fluorescence microscopy was performed. Changes in cell membrane lipid order between the monomer gel/liquid-ordered phase (fluorescent at 430 ∼ 470 nm) and the excimer liquid phase (fluorescent at 480 ∼ 550 nm) were measured by confocal microscopy.

Preparation of sarkosyl-insoluble PHF-tau from tg rat brain

Purified sarkosyl-insoluble PHF-tau from Tg rat brains was isolated according to a previously published protocol [22]. For isolation of sarkosyl-insoluble tau, we used brain tissue from 4, 6, 8, 10, 12, and 14-month-old Tg animals (n = 6). Briefly, 20 mg of the medulla and pons from a Tg rat brain enriched in PHF-tau were dissected, cleaned of blood vessels and meninges, and used as a starting material. The brain tissue was homogenized on ice in 10 volumes of ice-cold SL buffer (20 mM Tris- HCl, pH 7.4, 800 mM NaCl, 1 mM EGTA, 1mM EDTA, 0.5% β-mercaptoethanol, 10% sucrose, 1x protease inhibitors Complete, EDTA-free). The homogenate was centrifuged for 20 min at 20,000xg. Solid sarkosyl (Sigma-Aldrich, St. Louis, MO, USA) was added to the supernatant to achieve a 1% concentration. The samples were stirred for 1 h at RT. The samples were spun for 1 h at RT in a Beckman ultracentrifuge using SW 120 Ti rotor (Beckman Coulter, Inc., Brea, CA, USA) at 100,000xg. After sarkosyl extraction and ultracentrifugation, pellets containing PHF-tau were dissolved in a 1 x SDS sample loading buffer and boiled for 5 min. Samples were determined by following western blot analysis.

Biochemical Western blot analysis

Samples were separated on 12% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane in 10 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS, pH 11, Roth, Karlsruhe, Germany). The membranes were blocked in 5% milk in Tris-buffered saline with 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) (TBS-T, 137 mM NaCl, 20 mM Tris-base, pH 7.4, 0.1% Tween 20) for 1 h and incubated with primary antibody overnight at 4 °C. As phospho-dependent anti-tau antibodies, we used: mouse monoclonal anti-rat pThr181 (1:1000, Invitrogen Life Technologies, Carlsbad, CA), mouse monoclonal anti-rat pSer202/pThr205 (AT8, 1:1000, Invitrogen Life Technologies, Carlsbad, CA), mouse monoclonal anti-rat pThr231 (1:1000, Invitrogen Life Technologies, Carlsbad, CA), mouse monoclonal anti-rat pThr181 (1:1000, Invitrogen Life Technologies, Carlsbad, CA), rabbit polyclonal anti-rat pSer199 (1:1000, Invitrogen Life Technologies, Carlsbad, CA), rabbit polyclonal anti-rat pSer262 (1:1000, Invitrogen Life Technologies, Carlsbad, CA), mouse monoclonal anti-rat DC217 (1:200, Axon Neuroscience R&D SE, Bratislava, Slovakia) and polyclonal anti-rat pThr212 (1:1000, Invitrogen Life Technologies, Carlsbad, CA). For total tau, we used a mouse monoclonal anti-rat DC25 antibody (recognizing epitope 368–376, 1:200, Axon Neuroscience R&D SE, Bratislava, Slovakia), GFAP (1:1000, Abcam, Cambridge, UK), IBA-1 (1:1000, FUJIFILM Wako, USA), AQP4 (1:500, Abcam, Cambridge, UK). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies in TBS-T (1:3000, Dako, Glostrup, Denmark) for 1 h at RT. Immunoreactive proteins were detected by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific, Pittsburgh, USA) and the signals were digitized by Image Reader LAS-3000 (FUJIFILM, Bratislava, Slovakia). The signal was semi-quantified by ImageJ software.

Quantitative analysis of immunocytochemical and immunohistological data

Relative staining patterns and intensity of projections from immunohistochemical and immunocytochemical stainings were visualized by confocal microscopy and evaluated. ImageJ (public domain ImageJ software) was used for the evaluation and quantification. We quantified 10 slices from each sample. For semiquantitative analysis, the color pictures were converted to grayscale 8-bit TIFF file format, and regions of interest were analyzed. The grayscale 8-bit images were converted to 1-bit images, on which the number of immunolabeled structures localized in the area of interest was measured. The average intensity/pixel values of each area were then calculated, and the average intensity/pixel values representing the background intensity were subtracted from those of the immunolabeled areas.

Cytokine determination by Bio-plex assay

100 mg of the brainstem from SHR-24 and control animals (8-month old) was homogenized in 1 ml of ice-cold PBS with protease inhibitors complete EDTA-free (Roche, Mannheim, Germany). The protein concentration of brain extracts was determined by Bio-Rad protein assay (Bio-Rad Laboratories GmbH, Germany). 600 µg of total proteins were loaded per well. The plasma and CSF were diluted according to the recommendation of the manufacturer. The level of cytokines was measured by Bio-Plex Pro rat cytokine 23-plex assay (Bio-Rad Laboratories, California, USA) according to the manufacturer’s protocol. The Bio-PlexTM 200 system with Bio-Plex ManagerTM software version 6.1 (Bio-Rad Laboratories, Inc., CA, USA) was used to acquire and analyze the data.

Data analysis

Lipidomics and metabolomics data were processed and statistically evaluated in R software (www.r-project.org, 2019, v 3.5.0) using the Metabol package [23]. At first, the quality control-based locally estimated smoothing signal correction (LOESS) was applied to each dataset. The instrumental signal stability was monitored by internal standards across experimental and QC samples. The coefficient of variation (CV), based on QC samples, was calculated, and compounds (lipids or metabolites) with a CV higher than 30% were excluded from further processing. In the case of the brain, the correction of datasets to sample weight was carried out. The data were transformed by natural logarithm (ln), and the mean centering was applied. Bonferroni correction of p-values was applied to prevent false positivity of the t-test. Univariate and multivariate statistical methods were used to evaluate and visualize the results. Cytoscape software v3.8.2 was used to create metabolic and lipid maps according to the results of statistical analyses [24]. For metabolomics data, an Enrichment pathway analysis was performed using the web-based platform MetaboAnalyst (v5.0) [25] using our in-house database, which contained up to 53 metabolic pathways (Additional file Table S1). The lipid ontology (LION) enrichment analysis web application was used for the enrichment analysis of lipidomic data [26]. Data from western blot analysis, immunochemistry, ELISA, and FACS were statistically analysed using GraphPad Prism software v.8.0.1 (Inc., La Jolla, CA, USA).