Cytosolic PrP Can Participate in Prion-Mediated Toxicity

Abstract

Prion diseases are characterized by a conformational change in the normal host protein PrPC. While the majority of mature PrPC is tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, topological variants of this protein can arise during its biosynthesis. Here we have generated Drosophila transgenic for cytosolic ovine PrP in order to investigate its toxic potential in flies in the absence or presence of exogenous ovine prions. While cytosolic ovine PrP expressed in Drosophila was predominantly detergent insoluble and showed resistance to low concentrations of proteinase K, it was not overtly detrimental to the flies. However, Drosophila transgenic for cytosolic PrP expression exposed to classical or atypical scrapie prion inocula showed a faster decrease in locomotor activity than similar flies exposed to scrapie-free material. The susceptibility to classical scrapie inocula could be assessed in Drosophila transgenic for panneuronal expression of cytosolic PrP, whereas susceptibility to atypical scrapie required ubiquitous PrP expression. Significantly, the toxic phenotype induced by ovine scrapie in cytosolic PrP transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that while cytosolic PrP expression does not adversely affect Drosophila, this topological PrP variant can participate in the generation of transmissible scrapie-induced toxicity. These observations also show that PrP transgenic Drosophila are susceptible to classical and atypical scrapie prion strains and highlight the utility of this invertebrate host as a model of mammalian prion disease. IMPORTANCEDuring prion diseases, the host protein PrPC converts into an abnormal conformer, PrPSc, a process coupled to the generation of transmissible prions and neurotoxicity. While PrPC is principally a glycosylphosphatidylinositol-anchored membrane protein, the role of topological variants, such as cytosolic PrP, in prion-mediated toxicity and prion formation is undefined. Here we generated Drosophila transgenic for cytosolic PrP expression in order to investigate its toxic potential in the absence or presence of exogenous prions. Cytosolic ovine PrP expressed in Drosophila was not overtly detrimental to the flies. However, cytosolic PrP transgenic Drosophila exposed to ovine scrapie showed a toxic phenotype absent from similar flies exposed to scrapie-free material. Significantly, the scrapie-induced toxic phenotype in cytosolic transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that cytosolic PrP can participate in the generation of transmissible prion-induced toxicity and highlight the utility of Drosophila as a model of mammalian prion disease.

Drosophila models of prionopathies: insight into prion protein function, transmission, and neurotoxicity

“…But sustained translational repression can also be deleterious by limiting the availability of critical proteins. A recent paper demonstrated that overexpression of the eIF2α phosphatase GADD34 rescues prion-induced pathogenesis in mice, supporting the key role of the PERK-eIF2α pathway in PrD [41**]. In flies, PERK overexpression enhanced human PrP toxicity (Fig.…”

“…These results are suggestive of prion-mediated conversion of transgenic ovine PrP. Recent studies showed that the toxicity of membrane-bound and cytosolic, but not secreted, ovine PrP increased in the presence of sheep prions [38*,41], suggesting their ability to promote PrP conversion in Drosophila . Flies expressing cytosolic PrP showed the most dramatic increase in toxicity in the presence of prions in the absence of PK-resistant PrP.…”

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].…”

“…The UAS-PrP fly line w; M{VRQcyt-PrP, 3xP3-RFP.attP}ZH-51D that is transgenic for cytosolic expression of ovine V 136 R 154 Q 171 [VRQ(cyt)] was generated as recently described [28]. Cre-mediated removal of the red fluorescent protein (RFP) gene from the VRQ(GPI) and VRQ(cyt) PrP fly genome was performed by conventional fly crosses.…”

“…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.…”