Critical Roles Of N-terminus Of Prion Protein In The Pathogenesis Of Prion Disease

Conformational conversion,from cellular prion protein（PrP^C）into the misfolded isoform(PrP^Sc),is the central pathogenic event of prion disease,however the underlying mechanism remains elusive.The mature PrP^Cis post-translationally modified by N-linked glycans and tethered to the lipid raft region of cellular membranes via a glycosylphosphatidylinositol（GPI）anchor at its C-terminus.It is widely expressed and most abundant in neurons.PrP^C can interact with lipid components of cell membranes and this interaction alters the structure stability of PrP^C.Recent studies show that the proximal N-terminal charged cluster（residues 23-28,CC1）has a positive effect on accelerating the pathogenic process in prion disease;Transgenic mice expressing PrP^C without octapeptide repeats（residues 51-90,OR）region on PrP-null background display a polarized pathogenesis when infected by different strains of prions,for example,RML-prion accelerates,but BSE-prion delays the disease process in these mice.To elucidate the influence of N-terminus of prion protein（PrP）on conformational conversion and disease pathogenesis,we systemically studied CC1 and OR regions with both in vitro and in vivo approaches as follows:First,we generated two recombinant PrP^C（rec PrP^C）mutants:the Met4-1 andΔN6.In the Met4-1,all amino acids with positive charges in the CC1 region were replaced by the neutral methionine residue,whereas and in theΔN6,amino acid sequence of CC1 region was completely deleted.We found that both Met4-1 andΔN6 can form the protease K（PK）-resistant conformer（r PrP-res）,although the level of convertability of these two mutants was significantly lower than that of WT rec PrP^C.All rec PrP^Sc（WT,Met4-1,ΔN6）caused 100%mortality of C57BL/6j mice,however mice infected byΔN6-rec PrP^Sc had significant longer lifespan,but those by Met4-1-rec PrP^Sc had shortest lifespan when compared to WT-rec PrP^Sc.This divergent outcome is likely due to a distinct neuron toxicity of different rec PrP^Scconformers,which is consistent with the different neuropathological lesion profiles in these mice.Second,we analyzed the interaction between CC1 region and anionic lipid（POPG）in vitro and our data showed that an enhanced electrostatic force predominantly affects CC1 region and lipid interaction,which favors the PrP conformational conversion.All data of CC1 region collectively revealed that CC1 greatly impacts PrP-lipid interaction,PrP conformational conversion,and the resulting PrP^Sc conformation,which leads to the differences in neuropathological lesions.Furthermore,we generated the rec PrP^C mutant,ΔOR,in which the amino acids located in OR region are completely deleted.Results showed that both WT-and ΔOR-rec PrP^C can convert into the similar r PrP-res conformer,and the level of convertability ofΔOR was significantly higher than that of WT rec PrP^C.Moreover,the self-bred offspring of Prnp-ΔOR^+/-mice constructed by CRISPR-Cas9 technique were intracerebrally inoculated with WT prion.We found that the amount of endogenous expression of PrP^C-ΔOR（WT<Heter<ΔOR）is correlated positively with the lifespan of inoculated mice,but negatively with the level of neuropathological lesion and brain PrP^Scaccumulation of inoculated mice.We then analyzed the interaction between OR region and anionic lipid（POPG）,and found that the reduction of hydrophobic force predominantly affects interaction between OR region and lipid,which disfavors the conformation conversion of PrP.Thus,the OR region may not be beneficial for the structure conversion of PrP,but plays roles in increasing toxicity of PrP^Scin vivo.Altogether,our study elucidate that CC1 and OR regions have distinct functions in the PrP^C-to-PrP^Scconversion and provide a new insight into the molecular mechanism of prion pathogenesis.