Activation of microglia by retroviral infection correlates with transient clearance of prions from the brain but does not change incubation time

Abstract

Prion diseases are fatal transmissible diseases, where conversion of the endogenous prion protein (PrP^C ) into a misfolded isoform (PrP^Sc ) leads to neurodegeneration. Microglia, the immune cells of the brain, are activated in neurodegenerative disorders including prion diseases; however, their impact on prion disease pathophysiology is unclear with both beneficial PrP^Sc -clearing and detrimental potentially neurotoxic effects. Moreover, monocytes entering the brain from the periphery during disease course might add to disease pathophysiology. Here, the degree of microglia activation in the brain of prion infected mice with and without an additional intraperitoneal retrovirus infection was studied. Peripheral murine retrovirus infection leads to activation of parenchymal microglia without recruitment of monocytes. This activation correlated with transient clearance or delay in accumulation of infectious prions specifically from the brain at early time points in the diseases course. Microglia expression profiling showed upregulation of genes involved in protein degradation coinciding with prion clearance. This enforces a concept where microglia act beneficial in prion disease if adequately activated. Once microglia activation has ceased, prion disease reemerges leading to disease kinetics undistinguishable from the situation in prion-only infected mice. This might be caused by the loss of microglial homeostatic function at clinical prion disease.

Keywords: clearance; microglia; microglia signature; monocytes; neurodegeneration; prion disease; prions; protein misfolding; retrovirus.

Figure 1

PrP^Sc amount is unchanged by retrovirus infection. A. Paradigm of the inoculation procedure with time points depicted for retrovirus/mock infection (postnatal day 5; P5) as well as prion/NBH (normal brain homogenate from CD1‐mice) injections (P21). The upper scale shows age of the mice in days, whereas the lower scale shows time points of and after prion infection (dpi; days post infection). The preclinical experimental time points post prion infection and the corresponding age of the mice are depicted, respectively. B. Using standard and NaPTA enhanced Western blots developed with antibody Pom1 we could not detect differences in PrP^Sc amounts at preclinical time points between brains of prion only and prion + virus infected mice. Asterisks (*) indicate samples that were also investigated in the tga20‐bioassays.

Figure 2

Infectious prions are efficiently cleared from the brain of retrovirus infected mice at early time points. A. Titers of prion infectivity as measured by bioassay in tga20‐mice are similar in spleens of mice after prion only or prion + virus infection. B. In animals infected with high dose (HD) prions, high prion titers are detectable in brains of prion only infected mice. In contrast, brains of prion + virus infected mice do not have any measurable titer of infectivity 30 days post infection. C. After infection with low dose (LD) prions, infectious prions are not detectable in the brain 30 days post prion infection. At 60 dpi, similar prion clearing effects upon virus‐infection were detectable. D. In cerebellum, we could detect comparable effects in the bioassay at 60 dpi after low dose infection. ***P < 0.0001.

Figure 3

Brain resident microglia cell numbers are increased upon MoMuLV‐infection. A. Representative immunohistochemical staining for B‐cells (B220), T‐cells (CD3) and astrocytes (GFAP) of brains from MoMuLV infected and mock control mice do not show any signs of astrogliosis or lymphocytic infiltration at P21 (Scale bar 20 µm). B. In contrast, numbers of Iba1‐positive microglia cells in hippocampus, thalamus and cerebellum of virus infected mice were increased at P21 (Scale bar 20 µm). C. Quantification of Iba1‐positive cells at different time points indicated a significantly increase at P21 but not at P51 or P170. Each graph represents mean microglia number of four mice each. *P = 0.0289. D. Double staining in brain tissue of virus‐infected and age matched mock control mice revealed increase in Iba1‐positive/P2ry12‐positive but not Iba1‐positive/P2ry12‐negative cells in virus‐infected mice (Scale bar 50 µm).

Figure 4

MoMuLV infection correlate with lysosomal gene expression in microglia. A. Significantly downregulated genes in microglia cells after MoMuLV‐infection compared with mock control as assessed by Ncounter nanostring analysis (n = 4/group) P < 0.05. B. Significantly upregulated genes in microglia cells after MoMuLV‐infection compared with mock control as assessed by Ncounter nanostring analysis included several genes involved in protein degradation (n = 4) P < 0.05. (Serpine2 P = 0.009; Usp2 P = 0.002; Ctsd P = 0.049; Ctss P = 0.041; Ctsl P = 0.021; Snx24 P = 0.013) C. Double staining with Iba1 (red) and the lysosomal marker Lamp1 (green) revealed increased presence of lysosomes in microglia 30 days after virus infection. (DAPI staining for nucleus in blue; Scale bar 10 µm). D. Quantification of Lamp1 positive staining within microglia cells confirmed significant upregulation of lysosome abundance after virus infection (60 cells from n = 3/group) P < 0.0001.

Figure 5

Survival and PrP^Sc deposition pattern is not changed upon MoMuLV infection. A. Survival curve of mice infected intraperitoneally with MoMuLV retrovirus and intracerebrally with high or low dose RML 5.0 prions. B. Representative western blots of terminally sick mice revealed no difference in PrP^Sc amount in brain tissue (20 µg total protein per sample). C. Quantification of PrP^Sc amount (arbitrary units) in high dose infected clinically sick mice revealed no significant differences between prion only and prion + virus brains P = 0.5839 (n = 5/group). D. Immunohistochemical staining for PrP^Sc showed prion‐disease typical deposition pattern regardless of MoMuLV infection. As expected, no PrP^Sc could be detected in mock infected controls and mice infected with MoMuLV only. (Scale bar 20 µm).

Figure 6

Microglial homeostatic P2ry12 protein expression is shut down in clinical prion disease. A. Representative immunohistochemical staining for Iba1 and P2ry12 of three different brain regions in clinical prion diseased vs. aged matched healthy NBH injected control mice. (Scale bar 50 µm). B. Quantification of Iba1‐positive vs. P2ry12‐positive cells confirmed a significantly loss of P2ry12 reactivity in thalamus, hippocampus and cerebellum in prion infected mice. One graph represents mean microglia number in 0.325 mm² brain tissue of three mice each/group *P = 0.0134, **P = 0.0085, ***P < 0.0001.