Expression of Toll-like receptors in the cerebellum during pathogenesis of prion disease

Abstract

Prion diseases, such as scrapie, entail the accumulation of disease-specific prion protein (PrP^Sc) within the brain. Toll-like receptors (TLRs) are crucial components of the pattern recognition system. They recognize pathogen-associated molecular patterns (PAMPs) and play a central role in orchestrating host innate immune responses. The expression levels of Toll-like receptors (TLRs) in the central nervous system (CNS) were not well-defined. To establish a model of prion diseases in BALB/C mice, the 22L strain was employed. The features of the 22L strain were analyzed, and the cerebellum exhibited severe pathological changes. TLR1-13 levels in the cerebellum were measured using quantitative polymerase chain reaction (qPCR) at time points of 60, 90, 120, and the final end point (145 days post-infection). During the pathogenesis, the expression levels of Toll-like receptors (TLRs) 1, 2, 7, 8, and 9 increased in a time-dependent manner. This trend mirrored the expression patterns of PrP^Sc (the pathological isoform of the prion protein) and glial fibrillary acidic protein. Notably, at the end point, TLR1-13 levels were significantly elevated. Protein level of TLR7 and TLR9 showed increasing at the end point of the 22L-infected mice. A deeper understanding of the increased Toll-like receptors (TLRs) in prion diseases could shed light on their role in initiating immune responses at various stages during pathogenesis. This insight is particularly relevant when considering TLRs as potential therapeutic targets for prion diseases.

Keywords: PrPSc; Toll-like receptor; central nervous system; innate immune; prion disease.

Figure 1

Coronal sections of the hippocampus, cerebrum, and cerebellum from 22L-infected at the end point and the control mice reveal distinct PrP^Sc deposition patterns. These sections were immunoassayed with the monoclonal 3H4 antibody and photographed at a magnification of × 400. Dark brown areas indicate the positions of PrP^Sc accumulation. Notably, the style of PrP^Sc deposition differed in 22L-infected BALB/C mice. Specifically: Plaque-like PrP^Sc deposits were observed in the cortex and hippocampus. Diffuse PrP^Sc deposits were present in the cerebellum. In contrast, control sections showed an absence of PrP^Sc detection.

Figure 2

Coronal sections of the Cortex, Hippocampus, and cerebellum from 22L-infected at the end point and control mice revealed distinct GFAP expression. These sections were immunoassayed with the monoclonal GFAP antibody and photographed at magnification of × 400. Dark brown areas indicated the positions of GFAP expression (arrow pointing). Notably, GFAP expression increased in in the brains of scrapie-affected mice, with the cerebellum showing the most intense staining.

Figure 3

Changes in the expression of TLR1-13 and GFAP in the cerebellum during the pathogenesis of prion disease were investigated at different time points (60, 90, 120, and 145 days post-infection, dpi). The results are summarized as follows: Subgraph (A): Levels of TLR1, TLR2, TLR7, TLR8, TLR9, and GFAP. Subgraph (B): Expression of TLR3, TLR4, TLR5, TLR6, TLR11, TLR12, and TLR13. The mRNA expression levels were quantified using quantitative real-time PCR analysis. The values (fold change) represent the proportion of expression levels of TLRs and GFAP in 22L-infected mice compared to those in control mice (mean ± standard deviation, n = 4). An asterisk (*) indicates that the expression of a specific gene was statistically significant (P < 0.05) when compared with the expressions at any dpi.

Figure 4

Detection of TLR1, TLR2, TLR7, TLR8, and TLR9 expression in the cerebellum of 22L-infected mice at the end point and controls, as revealed by Western Blotting. (A) The proteins were detected using antibodies specific to TLR1, TLR2, TLR7, TLR8 and TLR9, respectively. β-actin, was used as a control and detected with a corresponding antibody. (B) The histogram shows the expression levels of TLR1, TLR2, TLR7, TLR8 and TLR9, calculated by grayscale value relative to β-actin. The values are represented as the fold change in comparison to the Control mice, with normalization to 1, ^*P < 0.05. The experimental data reveal statistical significance for TLR7 and TLR9.

Figure 5

During the pathogenesis of 22L-infected mice, the expressions of PrP^Sc (abnormal isoform of the prion protein) and GFAP (glial fibrillary acidic protein) in the cerebellum increased at 60, 90, 120, and 145 days post-infection (dpi). Total protein was extracted from the cerebellums, and 50 μg of protein was loaded into each lane. Here are the details: (A) GFAP Expression: We detected GFAP expression using western blot analysis with a monoclonal mouse anti-GFAP antibody. β-actin served as the housekeeping protein reference. (B) PrP^Sc Level: The level of PrP^Sc was measured by western blot using a monoclonal mouse anti-SAF antibody. (C) Fold change analysis: we analyzed the fold change of GFAP and PrP^Sc expression across different dpi using SigmaPlot. Notably, both GFAP and PrP^Sc exhibited time-dependent changes during the pathogenesis of prion disease. Please note that these findings provide valuable insights into the molecular events associated with prion infection in the cerebellum.