Distinctive Toll-like Receptors Gene Expression and Glial Response in Different Brain Regions of Natural Scrapie

Abstract

Prion diseases are chronic and fatal neurodegenerative diseases characterized by the accumulation of disease-specific prion protein (PrP^Sc), spongiform changes, neuronal loss, and gliosis. Growing evidence shows that the neuroinflammatory response is a key component of prion diseases and contributes to neurodegeneration. Toll-like receptors (TLRs) have been proposed as important mediators of innate immune responses triggered in the central nervous system in other human neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. However, little is known about the role of TLRs in prion diseases, and their involvement in the neuropathology of natural scrapie has not been studied. We assessed the gene expression of ovine TLRs in four anatomically distinct brain regions in natural scrapie-infected sheep and evaluated the possible correlations between gene expression and the pathological hallmarks of prion disease. We observed significant changes in TLR expression in scrapie-infected sheep that correlate with the degree of spongiosis, PrP^Sc deposition, and gliosis in each of the regions studied. Remarkably, TLR4 was the only gene upregulated in all regions, regardless of the severity of neuropathology. In the hippocampus, we observed milder neuropathology associated with a distinct TLR gene expression profile and the presence of a peculiar microglial morphology, called rod microglia, described here for the first time in the brain of scrapie-infected sheep. The concurrence of these features suggests partial neuroprotection of the hippocampus. Finally, a comparison of the findings in naturallyinfected sheep versus an ovinized mouse model (tg338 mice) revealed distinct patterns of TLRgene expression.

Keywords: astrocytes; microglia; neuroinflammation; prion; scrapie; toll-like receptors.

Figure 1

Representative neuropathology and immunohistochemical assessment of spongiosis, PrP^Sc deposition, microgliosis, and astrogliosis in the CNS of control and scrapie-infected sheep in the medulla oblongata (Mo), thalamus (Th), hippocampus (Hc), and frontal cortex (Fc). Graphs depict semi-quantitative assessment values for each parameter analyzed. (A) Hematoxylin–eosin staining showing spongiosis changes in the CNS from scrapie-infected sheep, and an absence of spongiosis in control sheep. (B) Detection of PrP^Sc with L42 antibody in scrapie-infected sheep brain regions. No immunolabelling was detected in the control group. (C) Ionized calcium-binding adaptor molecule-1 (Iba1) immunostaining showing increased microglial reactivity in scrapie-infected versus control sheep. (D) Glial fibrillary acidic protein (GFAP) immunostaining displaying increased reactive astrogliosis in scrapie-infected sheep compared to control sheep. (E) Statistical comparisons of spongiosis, PrP^Sc deposition, Iba1, and GFAP intensity between brain regions. Scores range from 0 (negative) to 5 (maximum intensity). Significant differences in spongiosis, PrP^Sc, Iba1, and GFAP were determined using Student’s t-test or Mann–Whitney U test for normally and non-normally distributed data, respectively. Statistical differences between brain areas were determined using one-way analysis of variance (ANOVA) or Kruskal–Wallis test for normally and non-normally distributed data, respectively. * p < 0.05, ** p < 0.01. Scale bar = 200 µm.

Figure 2

Western blot detection of PrP^res in the CNS of control and natural scrapie-infected sheep. Equal protein quantities were loaded for adequate comparison, and immunoblots were run using SHA31 monoclonal antibody for analysis of samples from the (A) medulla oblongata, (B) thalamus, (C) hippocampus, and (D) frontal cortex from scrapie-infected sheep with incipient clinical signs (samples 1–5) and with advanced clinical signs (samples 6–13). Line 14 shows the negative control and line 15 a sample of non-infected sheep brain homogenate not subjected to pK digestion.

Figure 3

Distribution of neuropathological lesions (HE), PrP^Sc deposition, astrogliosis (GFAP), and microgliosis (Iba1), in four different hippocampal regions in scrapie-infected and control sheep: pyramidal cell layers CornuAmmonis (CA) 1, 3, and 4, and the stratum lacunosum-moleculare (SLM). Graphs depict semi-quantitative assessment values for each parameter analyzed. (A) Mild spongiform changes were observed in all hippocampal regions analyzed. (B) Intraneuronal PrP^Sc pattern (L42) in CA1, CA3, and CA4 and widespread fine granular PrP^Sc pattern in the SLM. (C) Increased Iba1 staining in scrapie-infected versus control sheep. Magnified images show some of the morphological differences observed. (D) Signs of moderate reactive astrogliosis in scrapie-infected sheep compared with controls. Scores range from 0 (negative) to 5 (maximum intensity). Significant differences were determined using Student’s t-test or Mann–Whitney U test for normally and non-normally distributed data, respectively. * p < 0.05, ** p < 0.01. Scale bar = 200 µm.

Figure 4

Thalamus sections from scrapie-infected (intracerebral inoculation) and age-matched tg338 control mice immunostained to assess spongiosis (hematoxylin–eosin), PrP^Sc deposition (R486 antibody), astrocytes (glial fibrillary acidic protein; GFAP), and microglia (ionized calcium-binding adaptor molecule-1; Iba1). (A,E) Severe spongiform lesions located mainly in the neuropil of scrapie-infected versus control mice. (B,F) Several PrP^Sc-positive neurons are evident in scrapie-infected mice, but absent from controls. (C,G) Activated microglia displaying more intense immunostaining and thick processes in scrapie-infected mice versus controls. (D,H) Increased astrogliosis with hypertrophic cell bodies and processes in scrapie-infected mice versus controls. Scores range from 0 (negative) to 5 (maximum intensity). Significant differences were determined using Student’s t-test or Mann–Whitney U test for normally and non-normally distributed data, respectively. * p < 0.05, ** p < 0.01. Scale bar = 200 µm.

Figure 5

Western blot detection of PrP^res accumulation in the CNS of clinically affected tg338 mice intracerebrally inoculated with scrapie compared with matched controls. Equivalent protein quantities were loaded for adequate comparison and immunoblots were run using SHA31 monoclonal antibody. Molecular-weight markers (kDa) are indicated on the left side of the immunoblot.

Figure 6

Gene expression of TLR 1-10, MyD88, Trif, and CD36 in the medulla oblongata (A), thalamus (B), frontal cortex (C), and hippocampus (D) of control and scrapie-infected sheep. Data are represented as the mean fold ± SEM increase with respect to control sheep (expressed as a score of 1). Mean scores were compared using the Student’s t-test or Mann–Whitney U test for normally and non-normally distributed data, respectively. # p < 0.1, * p < 0.05, ** p < 0.01.

Figure 7

Gene expression of the anti-inflammatory cytokines TGF-β and IL-10, and the proinflammatory cytokines TNF-α and IL-6, in the thalamus (A) and hippocampus (B) of control and naturally scrapie-infected sheep. Data are represented as the mean ± SEM fold change with respect to controls (expressed as a score of 1). Mean scores were compared using the Student’s t-test or Mann–Whitney U test for normally and non-normally distributed data, respectively. * p < 0.05, ** p < 0.01.

Figure 8

Gene expression of TLR1-7, TLR9, and MyD88 in the thalamus of clinically affected tg338 mice intracerebrally inoculated with scrapie-positive inoculum versus controls (scrapie-negative inoculum). Results are expressed as the mean ± SEM fold change with respect to controls (expressed as a score of 1). Mean scores were compared using the Student’s t-test or Mann–Whitney U test for normally and non-normally distributed data, respectively. # p < 0.1, ** p < 0.01.

Figure 9

TLR4 protein expression in the medulla oblongata (Mo), thalamus (Th), hippocampus (Hc), and frontal cortex (Fc) of scrapie-infected sheep versus control sheep (n = 4 per group). β-actin was used as a loading control. The graph depicts densitometry values and indicates a significant increase in TLR4 expression in all regions in scrapie-infected versus control sheep. Data are expressed as the mean ± SEM. Mean scores were compared using the Student’s t-test. * p < 0.05.