Sex effects in mouse prion disease incubation time

Abstract

Prion disease incubation time in mice is determined by many factors including PrP expression level, Prnp alleles, genetic background, prion strain and route of inoculation. Sex differences have been described in age of onset for vCJD and in disease duration for both vCJD and sporadic CJD and have also been shown in experimental models. The sex effects reported for mouse incubation times are often contradictory and detail only one strain of mice or prions, resulting in broad generalisations and a confusing picture. To clarify the effect of sex on prion disease incubation time in mice we have compared male and female transmission data from twelve different inbred lines of mice inoculated with at least two prion strains, representing both mouse-adapted scrapie and BSE. Our data show that sex can have a highly significant difference on incubation time. However, this is limited to particular mouse and prion strain combinations. No sex differences were seen in endogenous PrP(C) levels nor in the neuropathological markers of prion disease: PrP(Sc) distribution, spongiosis, neuronal loss and gliosis. These data suggest that when comparing incubation times between experimental groups, such as testing the effects of modifier genes or therapeutics, single sex groups should be used.

Figure 1. C57BL/6 and FVB/N survival curves.

Survival curves for prion transmissions to C57BL/6J (A–D) and FVB/NHsd (E–H) mice. A and E, Chandler/RML prions; B and F, mouse passaged BSE prions; C and G, ME7 prions; D and H, MRC2 prions. % survival is shown on the y-axis and days post inoculation is shown on the x-axis. All data are significant (P<0.05) except for mouse-passaged BSE transmission to FVB/NHsd. Detailed values are given in Table 1.

Figure 2. PrP^C levels for male and female C57BL/6 and FVB/N mice.

PrP^C levels (ug/ml) were determined in triplicate using 10% (weight/volume) brain homogenate for male (n = 3) and female (n = 3) mice of each strain in a PrP specific ELISA. Data are shown normalised by total protein content (ug/ml and ×1000) as determined by a BCA assay (mean ± standard deviation). No significant differences were seen between males and females or between strains.

Figure 3. Histological features of Chandler/RML inoculated male and female FVB/N mice.

Comparison of histological features between male FVB/N (left panel, A–D) and female FVB/N mice (right panel, E–H) inoculated with Chandler/RML prions. Panels A and E show the PrP^Sc distribution in a cross section of the brain and panels B–D and F–H are higher power views of the hypothalamus (boxed area). (B and F) mild spongiosis (C and G) synaptic deposition of PrP^Sc (D and H) gliosis. Overall, the pattern of PrP^sc distribution, spongiosis or gliosis, shows no difference between both groups. Scale bar corresponds to 2 mm (A, E), 80 µm (B, F) or 160 µm in all other panels.

Figure 4. PrP^Sc distribution in the brains of C57BL/6 and FVB/N mice.

Comparison of prion protein staining between male (A, C, E, G) and female (B, D, F, H) mice inoculated with ME7 prions (A–D) and MRC2 prions (E–H). A, B and E, F represent C57BL/6 mice and C, D and G, H represent FVB/N mice. Overall, the pattern of PrP^Sc distribution shows no difference between male and females and is characteristic of the prion strain used. Scale bar corresponds to 2.5 mm for all panels except for C and D where it corresponds to 3 mm.

Figure 5. Western blot of PrP^Sc from the brains of C57BL/6 and FVB/N mice following Chandler/RML transmission.

Western blot of proteinase-K treated 10% w/v brain homogenates (n = 3 for both males and females) immunoblotted with anti-PrP monoclonal antibody ICSM-35 (D-Gen Ltd, UK). (A) C57BL/6 mice (B) FVB mice. The PrP^Sc from both male and female brains is characteristic of the RML scrapie prion strain and no sex differences are seen.