Cells persistently infected with prions continuously produce protease-resistant prion protein (PrPres). Here, we show that the PrP-res level in prion-infected Neuro2a (N2a) neuroblastoma cells decreased to 50 % of their initial level over the first 48 h and then recovered by 96 h after seeding. The level of cellular prion protein (PrP C ) also appeared to fluctuate, but did not influence the fluctuation of the PrP-res level. Prion-infected N2a cells, co-cultured with a higher number of prion-unsusceptible cells, had twice as much PrP-res as those cultured without unsusceptible cells, suggesting that cell density influences the fluctuation of PrP-res as. Direct cell-to-cell contact between cells, rather than soluble factors, was involved in the cell density-dependent increase in the PrP-res level. The cholesterol content, which is known to influence PrP-res formation, also changed depending on cell density. Our data suggest that alterations in cellular microenvironments controlled by cell density influence PrP-res formation.The causative agent of prion diseases, prion, is believed to be composed mainly of an abnormal isoform of prion protein (PrP Sc ). During the progression of the disease, a host-encoded cellular prion protein (PrP C ) undergoes a conformational change to form PrP Sc , which accumulates in the central nervous system. It is known that PrP Sc is composed of protease-sensitive and -resistant forms (Tzaban et al., 2002;Safar et al., 2005; Pastrana et al., 2006). The PrP Sc form that is resistant to limited proteolysis is referred to as PrP-res, and PrP-res is used as a diagnostic marker of prion diseases.Studies using prion-infected cells have provided many insights into prion biology, including the mechanism of PrP biosynthesis and the cell biological mechanism of prion propagation (Caughey & Raymond, 1991;Borchelt et al., 1992; Vey et al., 1996;Naslavsky et al., 1997;Marijanovic et al., 2009), and transmission of prions (Fevrier et al., 2004;Gousset et al., 2009;Mattei et al., 2009). Although cells persistently infected with prions produce PrP-res and possess prion infectivity, the PrP-res level is known to change depending on the culture conditions, such as composition of the medium and differentiation state of cells (Rubenstein et al., 1990;Bate et al., 2004). The PrP-res level also changes during routine cell passage, possibly caused by subtle differences in the culture conditions. PrPSc in prion-infected cells is believed to exist in a dynamic equilibrium between synthesis and degradation.Theoretically, after each cell division, the amount of PrP Sc per cell must halve, after which it increases again to an appropriate level until the next cell division (Weissmann, 2004). Indeed, a decrease in PrP-res levels in cultured cells 48-72 h after passage followed by recovery of the PrP-res level thereafter has been reported (Ghaemmaghami et al., 2007). During this process, certain host factors can modulate the equilibrium level of PrP Sc by changing the rate of PrP-res formation, although the factor...

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ABSTRACT. The selection of sheep with scrapie-resistant PrP genotypes is one of the control measures for transmissible spongiform encephalopathies in ruminants. In this study, we investigated the frequencies of PrP genotypes in meat breeds in Japan. The nationwide surveillance revealed that nearly half of the Suffolk sheep, a major meat breed in Japan, carried scrapie-susceptible AQ/AQ and AQ/VQ genotypes. In addition, the VQ haplotype, which confers high susceptibility to scrapie within sheep, was also found in Poll Dorset sheep. A trial of selective breeding using sires with scrapie-resistant PrP genotypes AQ/AR and AR/AR could raise the ratio of scrapie-resistant sheep from less than 50% to 80% within 3 years. However, the use of sires with the AR/AR genotype and the selection of ewes would be required to achieve a higher ratio of scrapie-resistant sheep. KEY WORDS: prion, scrapie, transmissible spongiform encephalopathy.

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