Prion-induced and spontaneous formation of transmissible toxicity in PrP transgenic Drosophila

Abstract

Prion diseases are fatal transmissible neurodegenerative diseases of various mammalian species. Central to these conditions is the conversion of the normal host prion protein PrP(C) into the abnormal prion conformer PrP(Sc). Mature PrP(C) is attached to the plasma membrane by a glycosylphosphatidylinositol anchor, whereas during biosynthesis and metabolism cytosolic and secreted forms of the protein may arise. The role of topological PrP(C) variants in the mechanism of prion formation and prion-induced neurotoxicity during prion disease remains undefined. In the present study we investigated whether Drosophila transgenic for ovine PrP targeted to the plasma membrane, to the cytosol or for secretion, could produce transmissible toxicity following exposure to exogenous ovine prions. Although all three topological variants of PrP were efficiently expressed in Drosophila, cytosolic PrP was conformationally distinct and required denaturation before recognition by immunobiochemical methods. Adult Drosophila transgenic for pan neuronally expressed ovine PrP targeted to the plasma membrane, to the cytosol or for secretion exhibited a decreased locomotor activity after exposure at the larval stage to ovine prions. Proteinase K-resistant PrP(Sc) was detected by protein misfolding cyclic amplification in prion-exposed Drosophila transgenic for membrane-targeted PrP. Significantly, head homogenate from all three variants of prion-exposed PrP transgenic Drosophila induced a decreased locomotor activity when transmitted to PrP recipient flies. Drosophila transgenic for PrP targeted for secretion exhibited a spontaneous locomotor defect in the absence of prion exposure that was transmissible in PrP transgenic flies. Our data are consistent with the formation of transmissible prions in PrP transgenic Drosophila.

Bioassay of prion-infected blood plasma in PrP transgenic Drosophila

“…To achieve this, we have used flies that express either GPI-anchored ovine V 136 R 154 Q 171 [VRQ(GPI)] PrP [25,26] or cytosolic ovine V 136 R 154 Q 171 [VRQ(cyt)] PrP [28]. We have already established that both PrP transgenic fly lines are susceptible to ovine scrapie prion infectivity, which does not induce neurotoxicity in non-transgenic 51D Drosophila [25,26,28].…”

“…PK-resistant PrPSc in the form of PrP27-30 was detected following protein misfolding cyclic amplification (PMCA) using head homogenate from ovine prion-exposed VRQ(GPI) PrP transgenic Drosophila. This was not due to carry over of PG127-infected brain material at the time of exposure of the flies to sheep scrapie since PrP27-30 was not detected using head homogenate from similarly treated non-transgenic 51D flies [26].…”

“…Clinical prion disease was not detected in tg338 mice inoculated with head homogenate from scrapie-exposed VRQ(cyt) Drosophila (data not shown). However, our previous studies have shown that head homogenate from scrapie brain-exposed VRQ (cyt) PrP transgenic Drosophila can induce neurotoxicity in recipient PrP transgenic flies during fly-to-fly transmission studies [26].…”

“…We have previously shown that ovine PrP transgenic Drosophila accumulate disease-associated PrP after exposure to ovine prions [26]. PK-resistant PrPSc in the form of PrP27-30 was detected following protein misfolding cyclic amplification (PMCA) using head homogenate from ovine prion-exposed VRQ(GPI) PrP transgenic Drosophila.…”

“…We have previously generated Drosophila transgenic for ovine PrP and have shown that after exposure to exogenous ovine prions at the larval stage these flies develop a neurotoxic phenotype in adulthood, evidenced by decreased locomotor ability and the accumulation of Proteinase K (PK)-resistant PrPSc [26][27][28]. Here, we determined the sensitivity of ovine PrP transgenic Drosophila to prion infectivity by exposure of these flies to a dilution series of scrapie-infected sheep brain homogenate.…”