Investigating The Molecular Mechanism Of Prion Species Barrier And Prion Infected Mouse Model By Orally Administered Recombinant PrP^Sc

Prion diseases,also called as transmissible spongiform encephalopathies,are a group of zoonotic fatal neurodegenerative diseases,which can be sporadic,inherited or acquired.Unlike typical viruses,the infectious agent of prion diseases is a misfolding isoform of celluar prion protein(PrP^C)that can induce PrP^C convert into this misfolding conformation(PrP^Sc),leading to PrP^Sc propagation in the host.This infectious PrP^Sc is designated as prion.The genetic information of prion is stored in its conformation,which is unlike typical viruses.Serial protein misfolding cyclic amplification?s PMCA?has been widely used to propagate prions in vitro.In the process of s PMCA,a small amount of PrP^Sc is added as seed into the uninfected brain homogenate or recombinant Pr P for co-incubation,which can induce PrP^C conversion into PrP^Sc.The newly formed PrP^Sc aggregates form a smaller oligomer core by sonication,providing new seeds for the subsequent conformation transition process.Our lab generated a prion with bacterially expressed mouse prion protein in the test tube with lipid and RNA as cofactors,which completely proved that genetic information of prion is stored in its conformation.We have finished the following work in this research.1.Investigating the molecular mechanism of prion species barrier.The species barrier of prion diseases is one of the major research interests in the prion field.Mouse and hamster are both rodents sharing similar prion protein primary sequence,with only 11 mismatched amino acids between them.However,the strong species barrier makes crossspecies prion transmission rather inefficient.We systematically compared the effect of the 11 mismatched amino acids on prion species barrier.We found that 3 of the mismatched amino acids?I138M,M204 I,54G?have high impact on prion propagation and that I138 M has a very strong dominant negative effect.2 of the mismatched amino acids?V111M and V214T?can decrease the effiency of prion propagation.We generated various combinations of mutations among these 5 sites on hamster Pr P.However,these combomutations are not sufficient to overcome the barrier for mouse prion to transmit to hamster Pr P.Some of them have rather strong dominant negative effect,while hamster Pr P??54GM111V-M138I-I204M-T214V?appear to propagate better in the presence of certain amount of mouse Pr P.Moreover,this mutant showed encouraging results in propagating the prion conformation to wild-type hamster Pr P.Whether this recombinant hamster PrP^Sc is infectious or not remains to be proved by animals experiments.2.Prion infected mouse model by orally administered recombinant PrP^Sc.After intracerebral injection of recombinant PrP^Sc,wild-type mice reached the terminal stage of disease in ¹80 days.Intraperitoneal infection with this prion results in terminal disease in wild-type mice in ²10days.In order to further understand the infection pathway of recombinant prion,we tested orally administered route to infect wild-type mice with this recombinant prion.After orally administered prions,wild-type mice reached the terminal stage of disease in ²96 days and the infection rate is about 10%-15%.Characterization of diseased mice revealed classic neuropathology of prion disease,confirming that these mice succumbed to prion disease.Although some of the wild-type mice succumbed to prion disease after oral exposure,most of these mice appeared to be asymptomatic and were euthanized at ⁶50 days.Serially transmit to wild-type mice resulted in clasping in most of these mice,indicating that there are animal individual differences in prion infection and that there are low-level of prions in the uninfected mice after oral exposure,which makes their brain homogenate pathogenic.Conclusion:?1?Each mismatched amino acid has drastically different impact on prion propagation.The strong dominant negative mutant opens a new avenue to develop novel agents to inhibit prion propagation.Meanwhile the dominant negative effect may make it possible to generate new prions in vitro.?2?Recombinant mouse prions can be transmitted through oral route,making wild-type mice succumbed to prion disease.These mice developed classic behavioral and pathological changes of prion disease,confirming that recombinant prion is sufficient to cause prion disease in wild-type mice via oral route.Even in mice that failed to develop clinical disease after oral exposure,low level of prions appeared to be generated in vivo and can be pathogenic.How to block the transmission of prions in nature is of great significance.