Prion diseases or transmissible spongiform encephalopathies are infectious neurodegenerative diseases of humans and animals. A major feature of prion diseases is the refolding and aggregation of a normal host protein, prion protein (PrP), into a disease-associated protease-resistant form (PrPres) which may contribute to brain damage. In FY12 we studied the generation of protease-resistant prion protein (PrPres) in a cell-free PMCA system. At each cycle of PMCA we quantitated the amount of prion infectivity produced and compared this to the amount of PrPres generated. The output of infectivity after 4 cycles was found to be 320-fold greater than the input, and was 3200 to 10,000 fold greater than input after 6 and 8 cycles respectively. These data showing generation of increasing amounts of infectivity in a cell-free system in the absence of ATP and divalent cations provided strong support for the hypothesis that the agent of prion infectivity is not a virus. However, the ratio of the infectivity titer to the amount of PrPres was lower in PMCA versus brain derived samples suggesting that a substantial amount of PrPres generated in vitro might be non-infectious. In other experiments in FY12 we studied the stimulation of cytokine release by cultured astroglia and microglia after stimulation by scrapie brain extracts. We previously reported that brain homogenates from prion-infected mice induced cytokine protein release in primary astroglial and microglial cell cultures. Here we measured cytokine release by cultured glial cells to determine what factors in infected brain contributed to activation of microglia and astroglia. In assays analyzing cytokine release, glial cells were not stimulated in vitro by either PrPres purified from infected mouse brains or PrP amyloid fibrils produced in vitro. However, significant glial stimulation was induced by a heat labile protease-sensitive protein present in clarified scrapie brain homogenates lacking PrPres. Eight different cytokines were detected after scrapie brain stimulation of astroglia but only two were detected in similar experiments using microglia. In FY12 we also studied a transgenic mouse system where a naturally occurring variation of human PrP (N171S) was inserted into PrP of mice. Mice expressing this mutation were susceptible to mouse scrapie strains ME7 and 22L, but ninety percent of the mice were resistant to strains RML and 79A. The remaining 10% of mutant mice developed disease very late and had low levels of brain PrPres. Surprisingly this resistance was not seen in cell-free in vitro experiments using PMCA. This transgenic mouse system may be an example of the conformational selection model where the structure of some prion strains does not favor interactions with PrP molecules expressing certain polymorphisms.
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