Abstract
Prion diseases are neurodegenerative disorders caused by misfolding of the normal prion protein (PrP) into a pathogenic "scrapie" conformation. To better understand the cellular and molecular mechanisms that govern the conformational changes (conversion) of PrP, we compared the dynamics of PrP from mammals susceptible (hamster and mouse) and resistant (rabbit) to prion diseases in transgenic flies. We recently showed that hamster PrP induces spongiform degeneration and accumulates into highly aggregated, scrapie-like conformers in transgenic flies. We show now that rabbit PrP does not induce spongiform degeneration and does not convert into scrapie-like conformers. Surprisingly, mouse PrP induces weak neurodegeneration and accumulates small amounts of scrapie-like conformers. Thus, the expression of three highly conserved mammalian prion proteins in transgenic flies uncovered prominent differences in their conformational dynamics. How these properties are encoded in the amino acid sequence remains to be elucidated.
Highlights
PrPSc has been postulated as the causative agent in prion diseases, and it is composed of specific folding species of prion protein (PrP) that are replicated by autocatalytic mechanisms [7]
Spongiform Degeneration in PrP Flies—Spongiform degeneration is the neuropathological hallmark of transmissible spongiform encephalopathies, and we have shown before that overexpression of wild type HaPrP induces prominent vacuolar degeneration of brain neurons in 30 days [12]
mouse PrP (MoPrP) was mostly soluble in young flies but less than 50% was insoluble in 30-day-old flies (Fig. 6A)
Summary
Drosophila Stocks and Genetics—The transgenic flies carrying hamster PrP (UAS-HaPrP) were described previously [12]. For expression of the PrP constructs, homozygous females for the Gal strains were crossed with males bearing HaPrP-M6, MoPrP-P1, or RaPrP-F22. Histology—Flies expressing PrP throughout the brain under the control of the ubiquitous da-GAL4, along with control flies expressing LacZ, were collected at 1 and 30 days after eclosion. Immunofluorescence—For mushroom body analysis, we collected flies expressing PrP or LacZ under the control of OK107Gal at days 1 and 40. Tissue Homogenates and Western Blot—Depending on the experiment, 1–20 whole flies expressing the PrP constructs ubiquitously under the control of da-Gal were homogenized in 20 –50 l of PBS containing 150 mM NaCl, 1% Triton X-100, 4 mM EDTA, and Complete protease inhibitors (Roche Applied Science) using a motorized pestle. 36898 JOURNAL OF BIOLOGICAL CHEMISTRY cipitated proteins were detected by Western blot with a mixture of 3F4 and 6H4 antibodies against the same membrane
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