Role Of Copper Ions In Prion Protein Aggregation And Pathology

Prion is a kind of hydrophobic protein without imunity, which can infect human and animals and replicate in host cells. All of the prion diseases are accepted to share a common pathogenic mechanism, which based on the conversion of normal highα-helix containing PrPc into infectious and pathogenic PrPSc, which is the highly stable andβ-sheet rich isoform [1-3].Accumulation of amyloid deposits in brains has been related to the prion diseases. It has been shown that aggregated amyloid fibrils derived from recombinant PrP in vitro caused disease in animal models [4]. Other than amyloid fibrils, the native state of PrPc is able to be converted into stable nonnative states with highβ-sheet content such as theβ-oligomer [5,6]. Also certain non-amyloidal oligomeric forms of PrP derived from PrPSc have been demonstrated toxicity [7]. Sufficient evidences accumulated over the last few years indicate that soluble oligomers are toxic species, which damage the cell functions in neurodegenerative diseases.Earlier in vitro experiments revealed that the PrP specifically binds Cu2+[8-10] and this led to the proposal that PrP may function as a cuproprotein in vivo. The four histidine-containing octarepeats region of the PrP located between residues 60 and 91 was found binding Cu2+ in a cooperative manner [11], while recent researches indicated that residues 96 and 111 play important roles in binding Cu2+[12-14]. Accumulating evidences demonstrated that copper was able to modulate the pathogenesis of prion disease.Our researches indicated that Cu2+ is a vital factor involved in the formation of PrP oligomers. Cu2+ triggered structural conversion of PrP (90-231) to P-sheet isoform was revealed by CD (circular dichroism) spectra and fluorescence measurement. Moreover, we obtained deposition of PrP at higher pH and isolated soluble oligomers by size exclusion chromatography at physiological acidic pH. AFM (atomic force microscope) images showed the morphology of oligomers with diameters of 39±7 nm. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Flow Cytometry (FCM) demonstrated that oligomeric PrP induced significant damage and apoptosis of S-KN-SH cells. Using confocal microscopic techniques we found that oligomer induces the aggregation of endogenetic PrP, which is compartmentalized into the lysosomes where it might trigger pro-apoptotic cell death signals. This indicates that at physiological acidic environment Cu2+ promotes the formation of soluble PrP oligomers, which are cytotoxic and induce the apoptosis of neuron cells.In addition, Cu2+ also modulates the PrP aggregation into amloidic fibrils. Cu2+ suppressed the fibration of PrP in physiological conditions. Scattering experiment showed that Cu2+ promoted the formation of larger PrP particles. But the fluorescence of ThT demonstrated that only in the neutral pH 7.0 condition free of Cu2+, PrP aggregated into amyloidic fibrils. While the presence of Cu2+ restrained the formation of PrP fibrils in the same condition. However in other weakly acidic conditions, the fluorescence of ThT did not increase, which indicated that the there was no PrP amyloidic fibrils. AFM confirmed the existence of PrP amyloid fibrils and showed the morphology with a diameter of about 15 nm and a length of about 300nm.Our researches confirmed that Cu2+is a key factor in PrP aggregation. One hand, it promotes the formation of toxic soluble oligomers; on the other hand, Cu2+ suppresses the fibration of PrP. These results may indicate that Cu2+ plays different roles in different phases of the PrP pathogenesis, which may be beneficial for the pathogenesis of prion and other protein construction diseases.