Cell viability assay
The Human Oligodendroglioma (HOG) cell line (Millipore, Burlington, MA, USA) is a clonal cancer cell line and originates from a surgically removed oligodendroglioma from a human patient. The cell line is described in more detail by Post and Dawson, and Buntinx et al. (48,49). The cells were cultured in DMEM media (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with 10% heat-inactivated FBS (Gibco) and maintained at 37°C 5% CO2.
Cell viability assays for HOG cells and primary oligodendrocytes were performed with Calcein AM Cell Viability Assay (R&D Systems, Minneapolis, Minnesota, USA). HOG cells were seeded into 96-well plates at a density of 8000 cells per well. After 1 day in vitro, cells were incubated for 24 h with either 56.4 mM DMSO (Sigma-Aldrich, Saint Louis, Missouri, USA) (volume corresponding to that of 10 µM 27-OH treatment), 282 mM DMSO (volume corresponding to that of 50 µM 27-OH treatment), 1 µM, 10 µM, or 50 µM 27-OH (Avanti Polar Lipids, Alabaster, AL, USA). 27-OH was diluted in DMSO. Non-treated (data not shown) and 0.1 mM H2O2 treated cells were included as negative and positive controls, respectively. The assay was performed according to the manufacturer’s instructions using a 2 µM Calcein AM Working Solution. The fluorescence of the plates was read with BMG Fluostar Galaxy (BMG Labtechnologies, Offenburg, Germany) with an excitation filter of 492 nm and an emission filter of 520 nm, and results were processed with its software (version 4.31-0).
For cell viability assay in 2D primary oligodendrocytes, tissue was obtained from optic nerves of 12-days-old Sprague Dawley rats, as described previously (50). Cells were seeded into 24-well plates bearing 12-mm-diameter coverslips coated with poly-D-lysine (10 mg/ml) at a density of 1 × 104 cells per well. Cells were maintained at 37°C and 5% CO2 in a chemically defined medium (50). After 1 day in vitro, cultured oligodendrocytes were treated with increasing concentrations of DMSO or 27-OH (0.1–10 µM) for 24 h, as is indicated above. Cultured oligodendrocytes were incubated with Calcein-AM (Life Technologies, Carlsbad, CA, USA) at 1 µM and 37°C for 30 min in fresh culture medium and thereafter washed in pre-warmed 0.1 M PBS three times. Emitted fluorescence was measured by a Synergy HT (Biotek, Winooski, Vermont, USA) spectrophotometer using excitation wavelength at 485 nm and emission at 528 nm.
Primary 3D co-cultures
For 3D cell culturing C57/BLJ6 mice were used. Female mice were let to breed with males overnight to ensure accurate embryonic day (E) of the embryos used for primary cell cultures. At E16 – E17, the embryos were sacrificed by decapitation, heads of the embryos were collected and, kept in Hibernate™-E Medium (Gibco) until dissection of brains. Dissection was performed in EBSS solution (Life Technologies) and only dissected hippocampus and cortex were used for the co-culture set-up.
To extract the cells from the tissue, tissues were first incubated at 37°C in a 1:1 mix of solution A (0.0625% trypsinization solution [Life Technologies] in EBSS) and solution B (0.08 mg/ml DNAse I solution [Roche, Basel, Schweiz] in EBSS) for approximately 15 min gently moving. Trypsinization was stopped with 10% heat-inactivated FBS or culture media #1 containing 2% B27 supplement (Gibco), 1% N2 supplement (Gibco), 100 units/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific), in DMEM/F-12 + GlutaMAX (Gibco). After supernatant removal, the tissue was mechanically disintegrated by pipetting up and down in culture media. The supernatant containing the cells was collected and centrifuged at 500 x g for 5 min.
For immunocytochemistry, pellet was thereafter resuspended in a new culture media #2 containing a 1:1 mix of Neurobasal media and DMEM/F12 + GlutaMAX, 0.5% GlutaMAX supplement (Gibco), 2% B27, 1% N2, and 50 ng/ml NGF (Gibco). For 3D culture droplets, diluted cell suspension and Matrigel (Corning Life Science, Corning, NY, USA) were mixed 1:1 to reach droplet sizes of 50 µl. Droplets were cultured in individual dishes precoated with poly-D-lysine (MatTek, Ashland, MA, USA). Approximately 15,000 cells were seeded in each droplet. Droplets were let to settle for 1 h before adding 2 ml of the culture media #2. Cultures were treated at DIV1 with 1 µM 27-OH or 5.6 mM DMSO. Non-treated cultures were included as negative controls (data not shown). Two-thirds of media were replaced every four days until fixation and immunofluorescence (IF) analysis. Cultures were maintained at 37°C 5% CO2 and fixed at DIV20.
For the time-course study, the pellet was resuspended in culture media #1. 110,000–125,000 cells were seeded per 75 µl droplet with Matrigel in a ratio of 1:1. Droplets were let to settle for 1 h before adding 1 ml of the culture media #1. Cultures were treated at DIV1 with 1 µM 27-OH or 5.6 mM DMSO. Cultures were maintained at 37°C 5% CO2 until harvesting at DIV3, 5 or 10, by removing media, washing with PBS, and lysing with 300–350 µl RLT buffer (Qiagen, Hilden, Germany).
Immunofluorescence staining in 3D cultures
3D cell cultures were fixed with 2% methanol-free formaldehyde (Thermo Fisher Scientific) for 5 min at RT. Cultures were blocked and permeabilised with 0.3% Triton X-100 (Sigma-Aldrich), 5% BSA in PBS for 1 h at RT. Primary antibodies were diluted in the blocking/permeabilization buffer and incubated overnight at RT. Secondary antibodies were diluted in the same buffer and were incubated for 2 h at RT in dark. To observe mature oligodendrocytes and oligodendrocyte linage cells following primary antibodies were used: oligodendrocyte marker mouse anti-MBP 1:500 (ab62631, Abcam, Cambridge, UK), and oligodendrocyte linage marker goat anti-Olig2 1:20 (AF2418, R&D Systems). Secondary antibodies used were Alexa Fluor 633-conjugated goat anti-mouse 1:500 (A21052, Invitrogen, Thermo Fisher Scientific), Alexa Fluor 488-conjugated donkey anti-goat 1:1000 (A11055, Invitrogen), and DAPI 405nm 1:1000. After antibody incubations, coverslips were mounted on the cultures with 90% glycerol.
A Nikon Eclipse Ti spinning disk confocal microscope using 20X objective coupled to a camera with a pixel size of 6.45 µm was used to image the cultures, and images were processed with NIS-Element software (version 5.11.02, Nikon, Tokyo, Japan). The images were further analysed using Imaris x64 software (version 5.9.0, Oxford Instruments, Zurich, Switzerland) by creating surfaces of desired channels: DAPI alone represented the total number of cells, Olig2+ DAPI colocalization the cells that are of oligodendrocyte lineage, MBP− Olig2+ DAPI colocalization immature oligodendrocytes, and MBP+ Olig2+ DAPI colocalization represented the mature oligodendrocytes.
Quantitative PCR (qPCR)
RNA was isolated from 3D cultures using the RNeasy® Mini Kit (Qiagen), following the manufacturer’s protocol. The concentration and quality of the RNA were measured with a nanodrop spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific). Reverse transcription was performed in the S1000 Thermal cycler (Applied Biosystems, Thermo Fisher Scientific) to yield cDNA using a standardised protocol for high-capacity cDNA reverse transcription (Thermo Fisher Scientific).
Gene expression of Pdgfrα (Mm00440701_m1, Life Technologies), Cc1 (Mm00545872_m1, Life Technologies), Olig2 (Mm01210556_m1, Life Technologies), and Gapdh (4352339E, Life Technologies) in primary cells from the time-course experiment was analysed by qPCR according to a standardised protocol for TaqMan™ gene expression assay (Thermo Fisher Scientific), using the 7500 Fast Real-Time PCR System (Applied Biosystems). Copy numbers of Pdgfrα (platelet-derived growth factor receptor α) and Cc1 (adenomatous polyposis coli clone 1) mRNAs were adjusted by mRNA copy numbers of Olig2. As an internal control, mRNA copy numbers of Olig2 were adjusted by copy numbers of Gapdh. The relative values of mRNA copy numbers were compared to the numbers in DMSO-treated DIV3 cultures.
CYP27 transgenic mice
8-month-old female, human sterol 27-hydroxylase overexpressing, transgenic mice (Cyp27Tg) and their age-matched non-transgenic littermates (Tg−) underwent behavioural studies. We studied two separate cohorts: For Morris Water Maze we tested 10 transgenic and 10 non-transgenic mice and for Y-maze and Fear Conditioning 9 transgenic and 5 non-transgenic mice. Cyp27Tg mice overexpress human CYP27A1 and have higher amounts of 27-OH when compared to WT mice, described in more detail by Meir et al. and Ali et al. (46,47). 4 Tg − and 4 Cyp27Tg mouse brain samples were used for Western blotting of total brain homogenates. Myelin enriched fractions were retrieved from 4 Tg − and 7 Cyp27Tg mouse brains.
Behavioural tests
Morris Water Maze was performed as described by Maioli et al. (51). Each mouse was tested for four trials per day, for five consecutive days. Reference memory was evaluated on the sixth day (probe test).
Y-maze was performed as described by Eroli et al. (52). The fear conditioning test was performed as described by Eroli et al. (52), with the supplementation of the conditional stimulus of sound on day one. Mice were exposed to a 55 dB sound at 5000 Hz that lasted for 30 seconds and was followed by a mild foot shock (0.3 mA for 2 sec). The sound-shock pairing was repeated three times in total with a 50-sec interval between each one. Cue fear conditioning was performed on day three, where mice were let to explore the new surrounding for 2 min, whereafter the sound (55 dB at 5000 Hz) lasted for 2 minutes continuously. The rectangular-shaped chamber was replaced for a round-shaped chamber (20 cm diameter x 35 cm high), and the stainless-steel grid floor was replaced for a black flat surface. Instead of wiping the chamber with ethanol, the chambers were cleaned between each mouse with hypochlorous water (50% dil). Freezing behaviour was defined as the complete absence of mobility within the same area for 2 seconds or longer and was measured through TSE Multi Conditioning software. The freezing % recorded during the habituation phase of day 1 (as a measure of baseline freezing) was compared to freezing % of day 2 to evaluate the context memory. To assess the cue memory, we measured the freezing % on day 3 before and during the sound stimulus.
Deparaffinisation of mouse brain sections
Paraffin-embedded mouse coronal brain sections of adult Cyp27Tg mice and Tg − mice were used for Luxol fast blue (LFB) staining of myelin and IF staining of PDGFRα and CC1. For both IF and LFB, two sections from three different mice and genotype were deparaffinised by serial incubations in xylene or Histolab-Clear (HistoLab, Gothenburg, Sweden) and ethanol (EtOH) of decreasing concentrations (xylene or Histolab-Clear: 2x 10 min, 99.5% EtOH: 2x 5–10 min, 95% EtOH: 1x 3–5 min, 70% EtOH: 1x 3–5 min), after which they were washed in deionised water.
Luxol blue staining of mouse brain sections
For myelin staining, the Luxol Fast Blue Stain Kit (Abcam) was used. Brain sections were incubated in LFB for 2 h at 60°C. After incubation, slides were rinsed thoroughly with distilled H2O and thereafter dipped in lithium carbonate for differentiation. Differentiation was continued by dipping the slides into 70% alcohol reagent until the grey matter was colourless. Additional sections were incubated with cresyl echt violet for 1 min at RT to confirm that tissue morphology is maintained during LFB staining (images not shown). After incubation, slides were quickly rinsed with distilled H2O, and sections were dehydrated with three changes in 99.5% ethanol. Sections were mounted on coverslips with Vecta Mount mounting media. Images were acquired with Nikon Eclipse E800 light microscope, using a 10X objective, coupled to a Nikon DS-Ri2 camera and processed with NIS-Elements imaging software (version 4.30.00). Blinded analysis of LFB stained sections was performed using ImageJ 1.52p (53) as described previously by Underhill et al. 2011 and Khodanovich et al. 2017 (54,55). The mean intensity of each brain section was measured from corpus callosum and fimbria. Mean intensities of the red channel (IR) in ROIs (Fig. 4A) were measured from RGB images of LFB stained sections without cresyl echt violet stain. Similarly, the mean intensity of the background (IB) was measured in each image. Optical densities (%) of each region were calculated as \(100\times \left(1-\left[\text{I}\text{R/}\text{I}\text{B}\right]\right)\), resulting in higher myelin content with increasing value.
Immunofluorescence staining of mouse brain sections
Immunofluorescence (IF) for MBP was performed on free-floating sections. Slices were permeabilized and blocked in 4% normal goat serum (NGS), 0.1% Triton X-100 in PBS (blocking buffer) for 1 h and incubated overnight at 4°C with primary antibody against MBP (SMI 99, 1:1000; Biolegend, San Diego, CA, USA). Slices were washed in PBS and incubated with fluorophore-conjugated Alexa secondary antibody (1:500) in blocking buffer for 1 h at RT. Samples were mounted with Fluoromount‐G. Images were acquired with Zeiss AxioVision microscope using a 10X objective and analysis was carried out in 2–3 sections per subject. Images were taken with the same setting for all experiments and mean intensity values were quantified from corpus callosum and cortex with Image J software.
For IF of CC1 and PDGFRα in deparaffinised tissue, a heat-induced treatment (95°C for 5 min) in R-Universal buffer (Aptum, Southampton, UK) was performed on all sections for epitope recovery. The sections used for CC1 staining were washed with cold PBS and treated with ice-cold 100% EtOH for 10 min at − 20°C. This step was not performed on the sections used for PDGFRα staining. The sections were blocked for 1 h at RT in blocking buffer with 0.3% Triton X-100, 10% NGS (Vector Laboratories, Burlingame, CA, USA) in PBS. The sections were then incubated with mixtures of primary antibodies rat anti-PDGFRα 1:100 (562777, BD Biosciences, San Jose, CA, USA) or mouse anti-CC1 1:200 (OP80, Millipore) together with anti-NeuN (ABN78, Sigma-Aldrich) in incubation buffer (0.3% Triton X-100, 10% NGS, 1% BSA) overnight at 4°C. Sections were incubated with goat anti-rat Alexa Fluor 488 1:300 (A11006, Invitrogen) or Goat anti-mouse Alexa Fluor 488 1:300 (A28175, Invitrogen) and anti-NeuN and DAPI 1:1000 secondary antibodies for 2 h at RT and protected from light. Finally, the sections were treated with an autofluorescence eliminator reagent (Millipore) for 5 minutes to reduce autofluorescence and mounted with SlowFade™ Gold Antifade (Thermo Fisher Scientific). The stained tissue was analysed using a confocal microscope (Zeiss LSM-800 Airy system) with a 20X objective. Three pictures from each region, corpus callosum, cortex, and hippocampus, were captured from one section of each mouse using the Zen software (ZEISS Microscopy, Jena, Germany). Imaging settings were kept constant for each staining type. In a blinded experiment, the number of PDGFRα and CC1 positive cells were quantified for each region (total area of approximately 918 mm2/region) by one rater.
Preparation of myelin enriched fractions
Dissected brains were removed from meninges, choroid plexus, cerebellum, and the brainstem. Brains were minced in DMEM. Total brain homogenates were incubated with 2.5 mg/ml DNAse I and 2.5 mg/ml Trypsine/EDTA for 30 min–1 h at 37°C on an orbital shaker at 180 rpm for tissue digestion. Thereafter, tissue suspension was mixed with DMEM and centrifuged at 1000 x g for 10 min at 4°C. Pellet was then resuspended in 20% w/v BSA-DMEM and centrifuged 1000 x g for 20 min at 4°C. The upper layer (myelin) was collected using a pasteur pipette.
Immunoblotting of brain and cerebrospinal fluid
Brains were homogenated with a douncer in RIPA buffer (50 nM Tris pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NPO-40 in 0.1 M PBS) supplemented with protease inhibitor cocktails (Roche). Afterwards, they were sonicated for 25 cycles at 80% amplitude (Labsonic M, Sartorius, Göttingen, Germany), centrifuged for 10 minutes at 1,200 rpm 4°C, and the supernatants were collected. Total protein content was quantified through Bradford assay (Bio-Rad, Hercules, CA, USA) and all the samples were brought to the same concentration. Samples were mixed with 1X sample buffer (62.5 mM Tris ph 6.8, 10% glycerol, 2% SDS, 0.002% bromophenol blue, and 5.7% β-mercaptoethanol in dH20) and boiled at 95°C for 8 min.
For myelin enriched fractions, 25 µl of each sample was mixed with 25 µl of 2X sample buffer. This process was performed on ice to enhance the lysis process and avoid protein degradation. Samples were boiled at 95°C for 8 min. Consequently, they were centrifuged for 1 minute at 14,000 x g and the supernatants were collected. Then, total protein content was quantified through RC DC Protein Assay (Bio-Rad) and all the samples were brought to the same concentration. Finally, they were boiled again at 95°C for 8 minutes.
Brain homogenates (10 µg per sample), myelin enriched fractions (20 µg per sample) and CSF (11.25 µl) were size-separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 4–20% Criterion TGX Precast gels and transferred to nitrocellulose membranes using a Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in 5% BSA in Tris‐buffered saline/0.05% Tween‐20 (TBS‐T) and proteins detected by specific primary antibodies against MBP (SMI 99, 1:1000; Biolegend), PLP (MAB388, 1:1000, Millipore), MOG (MAB5680, 1:1000, Millipore), CNPase (C5922, Sigma-Aldrich), and β‐actin (A20066, 1:5000; Sigma‐Aldrich).
CSF sampling and clinical biomarkers
CSF samples were collected by standard lumbar puncture between the L3/L4 or L4/L5 intervertebral space using a 25-gauge needle. CSF was aliquoted in polypropylene tubes and stored at − 80°C until further analysis. CSF Aβ42, t-Tau, and p-Tau concentrations were measured on fresh samples with commercially available sandwich enzyme-linked immunosorbent assays (Innogenetics, Ghent, Belgium) according to standardized protocols in the clinic. 27-OH levels were quantified in cerebrospinal fluid using isotope dilution mass spectrometry as described previously (56).
Statistical analysis
Analyses were carried out using Prism (version 8.2.1, GraphPad, San Diego, CA, USA), except for CSF data which were analysed using Stata software (version IC 16.1, StataCorp LLC, College Station, Texas, USA). Results are expressed as mean values and standard deviations. Data obtained from cell and animal studies were analysed with a two-tailed t-test, Mann-Whitney two-tailed U-test, one-way ANOVA, or repeated-measures ANOVA when appropriate. In the case of ANOVA, adjusted p-values are reported. Normality of data was assessed with the Shapiro-Wilk test, and for CSF data with skewness and kurtosis tests for normality. Zero-skewness log transformation was applied to skewed variables. For analysis of associations of CSF data, four linear regression models were applied. In Model 1 adjustment was made for age and sex. In Model 2 adjustment was made for age, sex, and Aβ42. In Model 3 adjustment was made for age, sex, and t-Tau. In Model 4 adjustment was made for age, sex, and p-Tau. In all models, myelin proteins were considered dependent variables and 27-OH an independent variable. In all analyses, a p-value of < 0.05 was considered significant.