Sex Effect in Mouse and Human Prion Disease

MAJOR ARTICLE Sex Effect in Mouse and Human Prion Disease Corinne Loeuillet,1 Pierre-Yves Boelle,2 Catherine Lemaire-Vieille,1 Marie Baldazza,1 Philippe Naquet,3 Pierre Chambon,4 Marie-France Cesbron-Delauw,1 Alain-Jacques Valleron,2 Jean Gagnon,1 and Jean-Yves Cesbron1 1 Sex effect on the incubation period of variant Creutzfeldt-Jakob disease (vCJD) disease in human and ME-7 murine models was investigated. In the 167 vCJD cases reported in the United Kingdom as of January 2009, age at onset was significantly lower in female patients (by 2 years) than in male patients after stratification on birth cohort. In C57/Bl6N mice infected with ME-7 scrapie strain, incubation was shorter in female than in male mice. The incubation period increased in castrated male mice after intraperitoneal infection but not after intracerebral inoculation. In the absence of androgen receptors, the incubation period for prion disease increased after intraperitoneal inoculation. In ovariectomized or estrogen receptor a–defective female mice, no effect was observed on the incubation period of mouse prion disease. These results show that androgens influence the prion diseases incubation period after inoculation at a peripheral site. Transmissible spongiform encephalopathies are infectious fatal neurodegenerative disorders. They are caused by the autocatalytic misfolding of the host protein PrPc. These diseases include bovine spongiform encephalopathy (BSE) and the subsequent human variant Creutzfeldt-Jakob disease (vCJD) resulting from BSE contamination. Although exposure to BSE in the United Kingdom has been widespread, !180 patients had developed clinical vCJD by October 2009 [1]. The altogether limited number of vCJD cases is consistent with the predictions of an earlier epidemiological model [2]. The main assumption of this model was that the risk of acquiring vCJD exponentially decreased after childhood, which was consistent with the age distribution of vCJD cases. Refining this model showed Received 8 January 2010; accepted 8 March 2010; electronically published 1 July 2010 Potential conflicts of interest: none reported. Financial support: Region Rhône-Alpes, Cluster 10 (to L.A.P.M. ) and the Centre National de la Recherche Scientifique (limited research contract to C.L.). Reprints or correspondence: Pr Jean-Yves Cesbron, Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163, Université Joseph Fourier, BP 170, F-38042 Grenoble cedex 9, France (). The Journal of Infectious Diseases 2010; 202(4):648–654  2010 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2010/20204-0018$15.00 DOI: 10.1086/654818 648 • JID 2010:202 (15 August) • Loeuillet et al that the risk actually peaked during adolescence, a pattern that could not be explained by change in meat consumption with age alone, therefore pointing to agedependent susceptibility to the disease [3]. The most likely explanation for this strong age-susceptibility relationship during childhood and adolescence is hormonal. In this case, there could be a difference in age at onset or death according to sex, because hormonal changes begin earlier in girls than in boys. Therefore, we investigated whether there was a sex difference in age at onset in human vCJD, and we then used a mouse model to test the hypothesis on the possible role of sexual hormones on the risk of prion diseases. We found a sex effect in both vCJD disease and in mice infected with ME-7 scrapie strain. We also observed that male castrated mice and androgen receptor– deficient mice died later than control littermates, suggesting that androgens and its receptor are involved and influence the incubation period of mouse scrapie disease. METHODS Human epidemiological data. We analyzed the age at onset according to sex for the 167 vCJD cases reported to the UK vCJD unit as of January 2009, all of Laboratoire Adaptation et Pathogénie des Micro-organismes, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 5163–Université Joseph Fourier, Grenoble, 2Université Pierre et Marie Curie–Paris 6, Institut National de la Santé et de la Recherche Médicale (INSERM) U707, Paris, 3Centre d’Immunologie de Marseille-Luminy, INSERM-CNRS–Université de La Méditerranée, Marseille, and 4Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, INSERM U964, Université Louis Pasteur de Strasbourg, Strasbourg, France tone of the tail. The incubation period was taken as the time from inoculation to the euthanasia of the mice. Mice were monitored 3 times per week, beginning 2 months after inoculation. Tissue specimens were collected and frozen (⫺80C) for subsequent Western blot analysis. Western blot analysis. Brain homogenates were prepared in a lysis buffer (10% w/v) containing 150 mmol/L NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 50 mmol/L Tris-Hcl (pH, 7.5), 2 mmol/L EDTA, and 1 mmol/L protease inhibitors (pepstatin and leupeptin). After 20 min of incubation at 4C and 10 min of centrifugation at 3000g, supernatants were collected, and total protein concentration was measured by bicinchoninic acid protein assay (Uptima). For PrPSc detection, the equivalent of 1 mg of total protein was incubated with 20 mg/ mL Proteinase K (Roche) at 37C for 30 min. The reaction was stopped by adding 4 mmol/L phenylmethylsulfonyl fluoride (PMSF; Euromedex). Proteins were centrifuged for 1 h at 240,000g on a 10% sucrose cushion, the pellets were resuspended in reducing Laemmli buffer, boiled at 100C for 5 min, and one-third was loaded onto a 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. After 2transfer, the nitrocellulose membrane was blocked with 3% BSA in PBS-0.1% Tween 20 and subsequently incubated in 3% BSA in PBS-0.1% Tween 20 with the SHA31 primary antibody (0.32 mg/mL; a gift of J. Grassi, CEA, Gif-sur-Yvette) for 1 h, followed by goat anti-mouse HRP conjugate (1:10,000; Sigma Aldrich) for 1 h. Blots were developed by enhanced chemiluminescence (ECL, Uptima) and exposed on X-Ray films (Hyperfilm ECL; Amersham Pharmacia). A minimum of 3 mice from each group were analyzed by Western blot analysis. Statistical analysis in mice. Incubation period data were expressed as median and interquartile range (IQR). Differences in incubation periods were tested by the Mann Whitney U test. To confirm these results and to account for multiple testing, we also performed an analysis of variance that included the effects of sex, route of administration, and castration status, with the Tukey correction for subgroup comparisons. In all comparisons, the level of significance was set at .05. RESULTS Sex effect in patients with vCJD. Data were obtained from the 167 vCJD cases reported to the UK vCJD unit as of January 2009. Ninety-four patients (56%) were male and 73 (44%) were female. This sex ratio is not significantly different from 50% (P p .12). The median age at onset of (...truncated)