EFV was purchased from Toronto Research Chemicals Inc., Ontario, Canada. The PK and Pefabloc (protease inhibitor) were purchased from Roche, Germany. All other reagents/chemicals were purchased from Sigma-Aldrich, USA. The PrP monoclonal antibody (mAb) 4H11 used in this study to detect PrP has been previously described . The antibodies anti-Cyp46A1 (ab82814, Abcam, USA), anti-flotillin-1 (610,821, Mouse BD Transduction Laboratories) and anti-β-actin (Cell signaling) were used. Secondary antibody conjugated with peroxidase, goat anti-mouse HRP, was obtained from Jackson Immuno Research, USA.
Female FVB mice (6–8 weeks of age; Charles River Lab) and female C57BL/6 J mice (6–8 weeks of age; Jackson Lab) were used in this study. The mice were kept at 12-h (hr)/12-h light/dark cycle and maintained temperature at 23 °C, in an environment with 60 ± 10% humidity. The mice were allowed to access food and water ad libitum. All animal experiments described in this study were approved by the University of Calgary Health Sciences Animal Care Committee (approved protocols: AC14-0165, Bioassay 1; and AC18-0158, Bioassay) according to the guidelines issued by the Canadian Council for Animal Care and ARRIVE guidelines.
Animal bioassay I
After acclimatization, FVB mice were randomly divided into four groups of n = 4 and intracerebrally (i.c.) inoculated with brain homogenate (1% w/v in PBS; 20 μl) of non-prion infected mice or terminally sick mice infected with RML, 22-L, and ME-7, respectively. The i.c inoculation procedure was performed using 25-gauge disposable needles under anesthesia. Four mice each were euthanized at 75, 100, 125 DPI and at the terminal stage (> 150 DPI) of prion disease. Euthanasia was performed under anesthesia using 5% isoflurane by CO2 overdose. Terminal stage was reached when progressive clinical signs of advanced prion disease were observed.
Animal bioassay II (EFV treatment)
After acclimatization, C57BL/6 J mice were randomly assigned into five groups of n = 10 in each group:
RML + EFV (0 DPI-DW)
RML + EFV (30 DPI-DW)
RML + EFV (50 DPI-DW)
RML + EFV (30 DPI-IP)
All mice were inoculated under anesthesia i.c. into the right parietal lobe with brain homogenate (20 μl of 0.1%) from a RML-inoculated terminally sick mouse using a 25-gauge disposable hypodermic needle. The mice were closely monitored for 10 days for any detrimental effects after i.c. injection. EFV (0.09 mg/kg/day) treatment in drinking water was started on the day of inoculation (0 DPI-DW) or 30 and 50 dpi, according to previously established protocols [19, 41]. For the other post-treatment group, i.p. injection of EFV (0.05 mg/kg/day) was started at 30 DPI 3 times per week for 130 days. After 130 days EFV was delivered at concentration of 0.09 mg/kg/day in drinking water. EFV was dissolved in 0.01% DMSO and added to the drinking water. Every 3 days the EFV solution in drinking water was replaced with fresh EFV solutions and continued until the experimental endpoint. The mice were monitored initially weekly and daily when progressive clinical signs of prion disease were evident. At terminal stage of prion disease, mice were euthanized by CO2 overdose under anesthesia and survival times were recorded. Several mice across all groups were euthanized before they reached terminal prion disease for intercurrent diseases and were excluded from statistical analyses (Additional file 1: Table S1).
Cell lines and EFV treatment
The murine neuroblastoma cell line N2a was obtained from ATCC (CCL-131). Cell cultures were maintained in Opti-MEM Glutamax medium (GIBCO, USA) with 10% fetal bovine serum, and penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere. N2a-wt cells are stably transfected cells overexpressing murine PrP. N2a cells persistently infected with mouse-adapted scrapie strains 22L (N2a-22L) or RML (N2a-RML) prions were used in the study [25, 43]. N2a-RML represent a cell line stably overexpressing 3F4-tagged murine PrP , in addition to endogenous mouse PrP. CAD5 neuronal cells (a generous gift from Dr. Mahal, Scripps Research Institute, Florida; ) were persistently infected with RML or 22L prions (CAD5-RML, CAD5-22L) and cultured at 37 °C in a 5% CO2 atmosphere in Opti-MEM Glutamax medium containing 10% bovine growth serum (Hyclone, USA) and penicillin/streptomycin. Cells were treated with EFV at different concentration (5 µM, 10 µM, 20 µM in DMSO) or with vehicle. After 72 h of EFV treatment cell lysates were collected and proceeded for analysis.
Cerebellar granular neuron (CGN) culture and EFV treatment
Newborn (7 days post-natal) C57BL/6 mice (Charles River, Saint Constant, Quebec, Ca) were used for preparing primary cultures of cerebellar granular neurons (CGN). CGN were mechanically extracted from the cerebella of newborn mice and enzymatically dissociated as previously described . Cells were plated at a density of 1.9 × 103 cells/mm2 on plastic culture wells pre-coated with 10 μg/ml poly-D-lysine. Cells were cultured in Dulbecco’s modified Eagle’s medium-Glutamax I high glucose (DMEM) (Life Technologies-Gibco, Ca) supplemented with penicillin and streptomycin (Life Technologies, Ca), 10% fetal bovine serum (Life Technologies, Ca), 20 mM KCl, and N2 and B27 neuronal supplements (Life Technologies, Ca). Cells were incubated at 37 °C in a humidified 5% CO2 atmosphere. Every week, the medium was supplemented with glucose (1 mg/ml) and antimitotics, uridine and fluorodeoxyuridine (10 μM) (Sigma-Aldrich) to reduce astrocyte proliferation in the culture.
After 48 h of seeding, CGNs were infected with RML mouse adapted scrapie prions. As previously described , RML-infected mouse brain homogenate (10% w/v in PBS) was sonicated and added at a final concentration of 0.01% to CGN cultures. After four days, the inoculum was removed, cultures were washed twice with media and fresh media was added for the rest of the experiment.
From 1- or 7-day post infection (dpi) as indicated, until the end of the experiment, infected cultures were treated every 4 days with various concentrations of EFV (5 and 10 µM), or with vehicle. Cells were lysed for PrPSc detection at different days post infection as indicated and lysates were processed for further analysis.
Cell lysis and proteinase K (PK) digestion
Cell lysis was done as described previously [46, 47]. For PK digestion, lysates were incubated with PK at a final concentration of 20 µg/ml (N2a/CAD5 cells) and 5 µg/ml (CGN) for 30 min at 37 °C. PK digestion was terminated by the addition of Pefabloc protease inhibitor. Proteins were precipitated by adding methanol and resuspended in sample buffer for immunoblot analysis.
Immunoblot analysis was performed as previously described . Protein samples were re-suspended in TNE buffer (50 mM Tris–HCl pH 7.5; 150 mM NaCl; 5 mM EDTA) and separated on 12.5% SDS-PAGE. Electroblotting was done using Amersham Hybond P 0.45 PVDF membranes (Amersham, USA). Membranes were incubated with primary and secondary antibodies as indicated and analyzed using Luminata Western Chemiluminescent HRP Substrates (Millipore, USA). The densitometric analysis of immunoblots was performed using ImageJ.
Preparation of brain homogenates
Brain homogenates (BH) were prepared in PBS (10% w/v) using a gentle MACS™ Dissociator for 2 min at room temperature, followed by centrifugation at 2,000 g for 1 min. The homogenates were aliquoted and stored at − 80 °C until further use. For immunoblotting, 10% BH was mixed with equal volume of cold lysis buffer (10 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 0.5% sodium deoxycholate) and incubated at 4 °C overnight. Resulting 5% BHs were incubated with either water for no PK or 50 µg/ml of PK (final concentration) for digestion at 37 °C for 1 h. PK digestion was stopped by addition of proteinase inhibitors (Pefabloc) to both no PK and PK-digested BHs. This was followed by addition of 3X sample loading buffer and boiling at 95 °C for 5–7 min. The samples were processed for immunoblotting as described above.
Immunofluorescence staining for Cyp46A1 in mouse brains
Paraffin-embedded mouse brain tissue samples (5 µm-thick sections) on gelatin-coated slides were deparaffinized three times with absolute xylene (5 min for each wash) and rehydrated with graded ethyl alcohol (100% to 70%). Then they were washed twice with TBST (10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20) for 10 min and incubated for 1 h with 2% normal goat serum as a blocking solution and 0.3% Triton X-100 in PBS. After blocking, the slides were incubated with primary antibody (rabbit-Cyp46A1 (ab82814) diluted 1:100 in blocking solution overnight at 4 °C. After primary antibody incubation, the sections were washed twice for 5 min each and incubated for 1 hr with Alexa Fluor 488 goat anti-rabbit or Alexa FluorTM 555 goat anti-rabbit secondary antibodies (Jackson Immunoresearch) (1:100). Coverslips were mounted with Dako fluorescent mounting medium (Molecular Probe, Eugene, OR). The immunofluorescence images were captured at same conditions for all images using a confocal laser scanning microscope (Zeiss LSM 700 confocal microscope). Five images per section (tissue) were captured from each respective group. Confocal images were converted to tagged image file format (TIF). The quantification of the immunofluorescence intensity in the same region of the brain areas (cerebellum, medulla) in the TIF images for all groups was performed using ImageJ software. The background of TIF images was optimized according to the threshold intensity, and the immunofluorescence intensity was analyzed at specified threshold intensity for all groups at same conditions and was expressed as the relative integrated density between the groups.
Immunofluorescence staining in neuronal cells
The non-infected CAD5 and infected CAD5-RML and CAD5-22L and N2a-RML cells were seeded on coverslips in 12 or 24 well plates. N2a-RML cells were treated with EFV (20 µM) for 3 days for PrPSc staining. Control cells were treated with DMSO for 3 days. After reaching 70% confluence the cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature (RT) and washed three times with PBS. Cells were incubated for 1 h with 10% fetal bovine serum (FBS) as a blocking solution and 0.1% Triton X-100 in PBS. For PrPSc staining, cells were treated for 7 min with 6 M guanidine hydrochloride, washed 3 times with PBS and further incubated with primary antibody 4H11 or Cyp46A1 diluted 1:100 in blocking solution for 1 h at RT. After primary antibody incubation, cells were washed three times (5 min each) in PBS and further incubated with secondary antibody (Alexa FluorTM 555 goat anti-mouse secondary antibody, Invitrogen -1:500) for 1 h at RT. Nuclei were stained with DAPI for 10 min. Cells were washed with PBS and after final washes, coverslips were mounted on slides using Mounting Medium (PermaFluor™, Thermo fisher). Images were collected and processed using a confocal laser scanning microscope (Zeiss LSM 700 confocal microscope) and all the images were taken using the same conditions. Image analysis and quantification of the mean PrPSc intensity per nucleus was done using the Zen Desk 3.2 imaging software with the zone of influence (ZOI) method. For Cyp46A1 the number of original confocal images per well of the chamber slide was five per group and the images were converted into TIF images. The fluorescence intensity of the same region of TIF images for all groups was measured using ImageJ software (National Institutes of Health, Bethesda, MD). The TIF image background was optimized according to the threshold intensity and the immunofluorescence intensity at the same threshold intensity for all groups was analysed and was expressed as the relative integrated density of the samples relative to control cells.
Flotation assays for lipid raft isolation
Lipid rafts were isolated as described previously . Briefly, 3 × 107 N2a-WT cells treated for 3 days with EFV (20 µM) or not were solubilized in 400 μl cold lysis buffer (NaCl 150 mM, Tris–HCl pH 7.5 25 mM, EDTA 5 mM, and Triton-X 100 1%) and incubated on ice in the cold room for 30 min. The cell lysates were mixed with Nycodenz 70% in TNE (NaCl 150 mM, Tris–HCl pH 7.5 25 mM, EDTA 5 mM) to a final concentration of 35% Nycodenz and were overlaid by 200-μl fractions of Nycodenz solutions with concentrations of 25, 22.5, 20, 18, 15, 12, and 8%. After ultracentrifugation (200,000 g, 4 h, 4 °C, Beckmann TLS55 rotor), fractions were collected from the top to the bottom of the gradient and precipitated with methanol for immunoblot analysis.
Data and statistical analyses
Statistical analyses and histograms were produced using GraphPad Prism software (GraphPad 8, Software, USA). For statistical analysis of immunoblot signals two-tailed independent Student’s t-test for two groups or for multiple groups, one-way analysis of variance (ANOVA) followed by Turkey’s post hoc test or Dunnett’s multiple comparison test, as applicable, was used. Values are expressed as mean ± SEM. The graphical representation of survival times of animals was done using a Kaplan–Meier plot. The log rank test was used for statistical analysis of differences between groups in the survival plot with median and pairwise comparisons between control and treated group. Mice euthanized for reasons not related to prion disease were excluded from the analysis. Significance = *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.