Prion infection of mouse brain reveals multiple new upregulated genes involved in neuroinflammation or signal transduction

Abstract

Gliosis is often a preclinical pathological finding in neurodegenerative diseases, including prion diseases, but the mechanisms facilitating gliosis and neuronal damage in these diseases are not understood. To expand our knowledge of the neuroinflammatory response in prion diseases, we assessed the expression of key genes and proteins involved in the inflammatory response and signal transduction in mouse brain at various times after scrapie infection. In brains of scrapie-infected mice at pre- and postclinical stages, we identified 15 previously unreported differentially expressed genes related to inflammation or activation of the STAT signal transduction pathway. Levels for the majority of differentially expressed genes increased with time postinfection. In quantitative immunoblotting experiments of STAT proteins, STAT1α, phosphorylated-STAT1α (pSTAT1α), and pSTAT3 were increased between 94 and 131 days postinfection (p.i.) in brains of mice infected with strain 22L. Furthermore, a select group of STAT-associated genes was increased preclinically during scrapie infection, suggesting early activation of the STAT signal transduction pathway. Comparison of inflammatory markers between mice infected with scrapie strains 22L and RML indicated that the inflammatory responses and gene expression profiles in the brains were strikingly similar, even though these scrapie strains infect different brain regions. The endogenous interleukin-1 receptor antagonist (IL-1Ra), an inflammatory marker, was newly identified as increasing preclinically in our model and therefore might influence scrapie pathogenesis in vivo. However, in IL-1Ra-deficient or overexpressor transgenic mice inoculated with scrapie, neither loss nor overexpression of IL-1Ra demonstrated any observable effect on gliosis, protease-resistant prion protein (PrPres) formation, disease tempo, pathology, or expression of the inflammatory genes analyzed.

Importance: Prion infection leads to PrPres deposition, gliosis, and neuroinflammation in the central nervous system before signs of clinical illness. Using a scrapie mouse model of prion disease to assess various time points postinoculation, we identified 15 unreported genes that were increased in the brains of scrapie-infected mice and were associated with inflammation and/or JAK-STAT activation. Comparison of mice infected with two scrapie strains (22L and RML), which have dissimilar neuropathologies, indicated that the inflammatory responses and gene expression profiles in the brains were similar. Genes that increased prior to clinical signs might be involved in controlling scrapie infection or in facilitating damage to host tissues. We tested the possible role of the endogenous IL-1Ra, which was increased at 70 days p.i. In scrapie-infected mice deficient in or overexpressing IL-1Ra, there was no observable effect on gliosis, PrPres formation, disease tempo, pathology, or expression of inflammatory genes analyzed.

FIG 1

Detection of cytokines in brain tissue of C57BL/10 mice at various times after infection with scrapie strain 22L. Protein levels of 11 cytokines in the brains of scrapie-infected mice were measured by multiplex assay (IL-12p40, CCL4, CCL5, IL-1a, CXCL9, CXCL10, and soluble TNF-α) or by quantitative ELISA (CCL22, CXCL13, IL-1Ra, and OSM) at 70, 94, and 131 days p.i., as indicated. Each data point represents the result from an individual mouse, and the bar in each data set represents the mean. Black circles with gray bars indicate 22L scrapie-infected groups, and open circles with white bars represent mock-infected (M) groups. dpi, days postinfection. The error bars indicate 1 standard deviation from the mean. Statistical analysis was performed using the Student t test to compare infected and mock-infected results at a given time point. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 2

Immunoblot analysis of mouse brain homogenates demonstrated increased STAT1, pSTAT1, and pSTAT3 in the brains at various times after scrapie infection. Proteins (50 μg/lane) from brain homogenates of mock-infected and 22L-infected mice were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with the indicated anti-STAT1, anti-pSTAT1, or anti-actin antibody (A) or probed with the indicated anti-STAT3, anti-pSTAT3, or anti-actin antibody as a control (B). The immunoblots are for representative mice at each time point, and the molecular masses of (p)STAT1α, (p)STAT1β, and (p)STAT3 are indicated to the left of each immunoblot (in kDa). In panel A, the fold change measured by densitometry of detectable STAT1α (middle) and pSTAT1α(S727) (lower), normalized to actin and relative to mock controls, is shown in a bar graph, where each data point represents the result for an individual mouse and is plotted on a log₂ scale. In panel B, the fold change measured by densitometry of detectable STAT3 (middle) and pSTAT3(Y705) (lower), normalized to actin and relative to mock controls, is shown as a bar graph, where each data point represents the result for an individual mouse and is plotted on a log₂ scale. Statistical analysis was performed using the Student t test to compare infected to mock-infected controls at a given time point. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 3

Comparisons of cytokines in the brain tissue of C57BL/6 mice infected with either scrapie strain 22L or strain RML. Protein levels of 14 cytokines in the brains of scrapie-infected mice were measured by multiplex assay (IL-12p40, CCL2, CCL3, CCL5, GM-CSF, IL-1α, IL-1β, IL-13, CXCL1, CXCL9, and CXCL10) or by quantitative ELISA (CCL22, CXCL13, and IL-1Ra) as indicated. Each data point represents the result from an individual mouse, and the bar in each data set represents the mean. The error bars indicate 1 standard deviation from the mean. Statistical analysis was performed using the Student t test to compare infected to mock-infected groups at a given time point. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

FIG 4

Challenge of IL-1Ra-deficient and IL-1Ra-overexpressing mice with two different strains of scrapie. Survival curves of IL-1Ra-deficient (IL1rn−/−) relative to C57BL/6 littermate controls inoculated i.c. with scrapie strain 22L (A) or RML (B) indicated no difference in scrapie disease progression when the endogenous IL-1 receptor antagonist was absent. Survival curves of IL-1Ra-overexpressing (TgIL1rn) and C57BL/6 littermate controls inoculated i.c. with scrapie strain 22L (C) or RML (D) suggested that blocking IL-1 signaling through IL-1RI has no observable effect on scrapie pathogenesis. To ensure that IL-1Ra levels in the brains were absent in IL1rn^−/− mice but elevated in TgIL1rn mice, IL-1Ra amounts in brain tissue were quantified in age-matched mock-treated (M) and 22L- and RML-inoculated mice at the clinical endpoint (E).

FIG 5

Detection of gliosis, vacuolation, and PrPres formation in scrapie-infected mice. (A) Astrogliosis in 22L-infected and age-matched uninfected IL-1Ra-deficient (IL1rn^−/−) and IL-1-Ra-overexpressing (TgIl1rn) mice was assessed in representative mice by using anti-GFAP antibody in sagittal brain and thalamus sections. Immunohistochemical analysis showed the density of activated astrocytes in 22L-infected IL1rn^−/− mice was similar to that of infected TgIL1rn mice but differed from results in uninfected mice. Similar GFAP results were obtained with brain sections from mice inoculated with strain RML (data not shown) and when sections from mice inoculated with strain 22L or RML were stained with anti-IBA-1 to demonstrate microglial activation (data not shown). H&E staining of thalamus sections showed vacuolation associated with scrapie infection that was absent in uninfected mice. Vacuolation was similar for infected IL1rn^−/−, TgIL1rn, and C57BL/6 control mice. All thalamus sections stained with anti-GFAP or H&E are shown at the same magnification. Bar, 100 μm. (B) Immunoblot detection of PrPres in the brains of representative IL1rn^−/−, TgIL1rn, and C57BL/6 littermate control mice inoculated with scrapie strains 22L and RML. All samples were treated with proteinase K as described in Materials and Methods. Each lane was loaded with 0.36 mg whole-brain equivalent and probed with anti-PrP antibody D13. Protein mass markers (in kDa) are indicated to the left of the immunoblots.