Quantitative Mass Spectrometry Analysis of the Pathological PrP Allotypes Present in the Brain of gCJD Affected Individuals

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders characterized by the accumulation, principally in the central nervous system (CNS), of the abnormal form (PrPTSE) of a host encoded protein, the cellular prion protein (PrPC). In humans some forms of TSEs have a genetic etiology, as they are associated to mutations in the PrP gene: thus it is extremely important to elucidate the role played by the altered residue in the pathological conversion of the protein. Aim of our work was to deduce the involvement of the mutated residue in the molecular mechanisms underlying genetic TSE pathogenesis by means of a quantitative mass spectrometry analysis of the PrPTSE allotypes accumulated in the brains of genetic Creutzfeldt-Jakob disease (gCJD) affected individuals. We have integrated our previous data on the gCJD patient carrying the R208H mutation with those regarding V210I gCJD patients in order to verify if diverse mutations regulate in a different fashion the process of molecular pathogenesis. The LC/MS (liquid chromatography/mass spectrometry) protocol developed by us (Schininà M.E. et al., 2003. Pure Appl. Chem., 75: 317-323) has been implemented in order to set up, by means of appropriate synthetic deuterated (internal standards) and non-deuterated (calibrants) peptides, a quantitative analysis of the pathological PrP allotypes. In all cases the amount of the mutant allotype is higher than the wild-type counterpart: in the R208H subject the mutant/wild-type ratio has been again estimated and it was around 3/1, while in all the V210I individuals it was lower than 2/1. These results demonstrate that the presence of the mutation is partially involved in favouring the pathological conversion of PrP, but it is not the only factor implicated in the pathogenesis of TSEs as the ratio of the two allotypes does not reveal a significantly higher amount of the mutant allotype compared to the wild-type one. Further investigations are needed in order to elucidate the molecular mechanisms underlying the pathogenesis of genetic human TSEs.