Ethics statement
The use of animal samples in this study was approved by the Ethical Committee of the National Institute for Viral Disease Prevention and Control, China CDC, approved the use of animal samples in this study. The animal housing and experimental protocols adhered to complied with the Chinese Regulations for the Administration of Affairs Concerning Experimental Animals.
Brain samples of scrapie infected rodent models
The brain samples from C57BL/6 mice that were intracerebrally inoculated with mouse-adapted scrapie strains 139A and ME7, as well as brain samples from hamsters that were intracerebrally inoculated with hamster-adapted scrapie agent 263K were enrolled in this study [16]. Briefly, approximately 1 microliter of brain homogenates (1:10 dilution) from 139A- and ME7-infected mice, as well as 263K-infected hamsters were intracerebrally injected into 3-4-week-old animals under halothane anesthesia. The pathogenic features, neuropathological findings, and clinical manifestations of these prion-infected rodents have been described elsewhere [16, 17]. The average incubation periods for 139A-, and ME7-infected mice were 183.9 ± 23.1 and 184.2 ± 11.8 days, and that of 263K-infected hamsters was 66.7 ± 1.1 days, respectively. Age-matched healthy animals were used as controls in this study.
Preparation of brain homogenates
The brain homogenates were prepared according to a previously described protocol [3]. The whole brain tissues from both the infected rodents and the control group were washed three times in TBS (10 mM Tris-HCl, 133 mM NaCl, pH 7.4). Subsequently, 10% (w/v) brain homogenates were prepared using a cold lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM Tris, pH 7.5) supplemented with the protease inhibitor cocktail set III (Merck, 535140, USA). To remove tissue debris, low-speed centrifugation at 2000×g for 10 minutes was performed, and the resulting supernatants were collected for further study.
Preparation of brain tissue paraffin
The surgically excised brain tissues from different scrapie-infected experimental rodents and normal animals were fixed in 4% paraformaldehyde for a duration of 3 hours. After conventional gradient dehydration, the brain specimens underwent infiltration with xylene and subsequent immersion in molten paraffin at a temperature of 56°C until solidification. These preparations were then ready for further analysis.
Cell culture
The cell lines SMB-S15 and its normal partner SMB-PS were obtained from the Roslin Institute, UK. SMB-S15 was originally derived by culturing brain tissues from a mouse infected with the Chandler strain of scrapie. In this strain, persistent replication of the abnormal prion protein PrPSc occurs through cell passage. On the other hand, SMB-PS was established by treating SMB-S15 cells with prion-free pentose [5]. The BV2 microglia and SHSY5Y cell lines were maintained under controlled conditions. The cell lines were cultured in a humidified incubator at an appropriate temperature using Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 5% CO2.
Preparation of cell lysates
The cells were harvested and subjected to low-speed centrifugation at 500×g for 10 minutes. Subsequently, the resulting pellets were then resuspended in a cold lysis buffer (P0013B, Beyotime, China) supplemented with protease inhibitor cocktails set III (535140, Merck, USA), and kept on ice for 30 minutes. After incubation, the supernatants were collected and divided into aliquots for subsequent analysis after determining the protein concentration using the BCA method (71285-3, Merck, USA).
Cell transfection
Recombinant plasmids expressing wild-type PrP (pcDNA3.1-PrP-PG5) and cytoplasmic PrP (pcDNA3.1-CytoPrP) were constructed previously [18]. Logarithmic-phase cells were seeded in a 6-well plate and transfected after 24 hours. Approximately 2 µg of each plasmid DNA was transfected using LipofectamineTM 3000 (L3000150, Invitrogen, USA) according to the manufacturer's instructions. Cells were harvested at 48 hours post-transfection for subsequent experiments.
Western blots
Aliquots of brain homogenates or cell lysates were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation, followed by electro-transfer onto nitrocellulose membranes. Subsequently, the membranes were blocked with 5% (w/v) skimmed milk in 1× Tris-buffered saline containing 0.1% Tween 20 (TBST) at room temperature for 30 minutes. After blocking, the membranes were incubated with various primary antibodies against GAP43(1:1000, YT1849, Immunoway, USA), GAP43(1:1000, 8945S, Cell Signaling Technology, USA), p-GAP43(1:1000, AB5401, Sigma, USA), PrP (1:1000, 6D11, Santa Cruz, USA), β-actin (1:5000, TA-09, ZSGB-BIO, China), GAPDH (1:2000, ET1601-4, HUABIO, China), Calnexin (1:1000, MABF2067, Merk, USA) at 4°C overnight. Following TBST washes, the membranes were incubated with specific HRP-conjugated secondary antibodies and visualized using a commercial ECL kit. The ChemiDocTM XRSC Imager (Bio-Rad, USA) was employed to capture the images.
Co-Immunoprecipitation (Co-IP)
Approximately 1 ml of different cell lysates was combined with 4 µg of the corresponding capture antibody and incubated with 50 µl of Dynabeads®-coated protein G (10004D, Invitrogen, USA) at RT for 1–2 h. Subsequently, the mixture was further incubated overnight at 4°C. The immunocomplexes were then isolated using a magnet, washed five times with a wash buffer, and subjected to SDS-PAGE analysis. Finally, specific detection antibodies were employed for Western blot analysis.
Immunofluorescence assay (IFA)
The brain tissues from both normal and scrapie-infected rodents were fixed in a 10% buffered formalin solution. Subsequently, paraffin sections with thicknesses of 5µm and 2µm were routinely prepared. Following this, the brain sections were permeabilized with 0.3% Triton X-100 in PBS for 30 minutes and then blocked with normal goat serum for 1 h. After blocking, the sections were incubated with 1:200-diluted GAP43(8945S, Cell Signaling Technology, USA), 1:200-diluted anti-NeuN (MAB377, Merck, USA), 1:200-diluted anti-GFAP (GTX-34759, GeneTex, USA), and 1:200-diluted anti-Iba1 (SAB2702364, Merck, USA) in dilution solution (PBS with 2% BSA and 0.3% Triton X-100) at 4°C overnight. The following day, the sections were incubated 1 hour at 37°C with Alexa Fluor 488-labeled goat anti-rabbit (A11034, Invitrogen, USA) secondary antibody, diluted at 1:200, and Alexa Fluor 568-labeled goat anti-mouse (A11031, Invitrogen, USA) secondary antibody, also diluted at 1:200. After removing the secondary antibodies, a final concentration of 1 mg/ml of DAPI was applied to stain the nucleus at room temperature for 30 minutes. The slices were then sealed, and the targeted protein images were observed and analyzed using confocal microscopy (LEICA, TCS/SP8, Germany). The integral optical density (IOD) values for each field-specific fluorescence staining were recorded. The IOD values of the specific stainings were normalized relative to that of the DAPI-specific staining.
Immunohistochemical Staining (IHC)
The brain sections were subjected to antigen retrieval by heating in a sodium citrate buffer at 100°C for 30 minutes using a microwave. Subsequently, the sections were treated with 3% H2O2 and methanol to block endogenous peroxidase activity for 10 minutes at RT, followed by blocking with 5% BSA at RT for 15 minutes. The sections were then incubated overnight at 4°C with antibody GAP43(1:200, YT1849, Immunoway, USA), PrP (1:1000, 6D11, Santa Cruz, USA). Afterward, the sections were incubated with an HRP-conjugated goat anti-rabbit antibody (1:250 dilution, SV0002-12, Boster, China) at 37°C for 1 hour and subsequently incubated with 3,3'-diaminobenzidine (DAB, AR1000, Boster, China). The staining was observed, and the sections were counterstained with hematoxylin (AR0005, Boster, China) for 1 minute before being dehydrated, and mounted using conventional methods as described previously. Images were captured using an OLYMPUS BX41 microscope equipped with DP Controller software. Quantitative analysis was performed using Image-Pro Plus 6.0 software from Media Cybernetics.
Nissl Staining
The brain paraffin sections were subjected to Nissl staining using a 1% cresyl violet solution for 30 minutes. After a brief rinse in distilled water, the sections were differentiated in 95% ethanol for 30s to achieve optimal staining intensity [19]. Following dehydration, the sections were carefully mounted and observed under an Olympus BX51 microscope. Neurons were identified by the presence of Nissl bodies within the cells.
Quantitative real-time PCR (qPCR)
The Power SYBR Green PCR Master Mix (Applied Biosystems, USA) was used for real-time PCR on the ABI 7900HT Fast Sequence Detection System (Applied Biosystems, USA). Total RNA was extracted from SMB cells and the brain tissue of rodents using Trizol reagent (15596026, Gibco, USA), followed by first-strand cDNA synthesis with the reverse transcription system (11752050, Invitrogen, USA)according to manufacturer instructions. Specific primers were designed based on the mouse GAP43 and GAPDH gene sequences from GenBank (M25667.1 and M88109.1). The primer sequences for GAP43 were as follows: forward 5'-CAAGATGGTGTCAAGCCGGA-3' and reverse 5'-TCTCCACACCATCAGCAACG-3'. The primer sequences for GAPDH were as follows: forward 5′-TTTGCAGTGGCAAAGTGGAG-3′ and reverse 5′GATGGGCTTCCCGTTGATGA-3′. PCR amplification was performed in triplicate for a total of 40 cycles (95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 seconds). Relative quantification of target gene expression was determined using the comparative Ct method where Ct represents cycle number at which amplification reaches fixed threshold. The 2-ΔΔCt method was employed to calculate the relative expression levels of the target gene by subtracting relative Ct value of target gene from that of the GAPDH gene.
Statistical analysis
The immunoblotting images and the degree of co-localization in IFA were quantitatively analyzed using Image J software. IFA quantification was represented by the IOD, which is the sum of the reaction intensities of all selected objects within the field of view. The strength of correlation was assessed using Pearson’s correlation coefficient (r), with values indicating strong correlation (r > 0.8), moderate correlation (0.5 < r ≤ 0.8), weak correlation (0.3 < r ≤ 0.5), rare or no correlation (r ≤ 0.3). Fluorescence intensity was analyzed using Operetta system from PerkinElmer, USA. The data were presented as mean values ± SEM (standard error of the mean). Differences between groups were evaluated using two-tailed Student’s t-test and one-way ANOVA test, and p-values were denoted as ***(p < 0.001), **(p < 0.01), *(p < 0.05), or ns (not significant).