John Hopkins

Charmaine Love

Wilfred Goldmann

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases that are characterised by long incubation periods, protein aggregation and vacuolation. During TSE pathogenesis the normal, cellular prion protein, (PrPC), which is encoded by the gene PRNP, misfolds and accumulates as abnormal disease associated prion protein, (PrPSc) within the central nervous system. Variants of the Prion protein gene are associated with susceptibility to TSE disease. For example sheep scrapie disease is modulated by several PRNP alleles, with certain alleles carried by susceptible animals being different from those carried by resistant animals. The mechanisms linking PRNP genetics and disease is poorly understood but may involve protein sequence, PrPC expression levels, and possibly differences in protein processing. Post-translational modification of PrPC leads to specific cleavage (alpha cleavage) between amino acids 115/116 of ovine PrP, producing two fragments C1 and N1. Cleavage of PrP may occur as a protective mechanism, as a response to changes in the cellular environment or as a feature of an as yet unknown biological function. In the context of TSEs, alpha cleavage may inadvertently provide a protective role by reducing available PrPC protein for conversion into PrPSc, assuming that the C1 fragment would be an inefficient substrate for conversion, the opposite theory was also proposed. The former hypothesis became the focus of this present study, with the idea that total full-length PrPC, total C1 or the ratio between full-length PrPC and C1 may be linked to differences in scrapie susceptibility. To investigate these aims the C1 fragment was measured as a percentage of total PrPC in different PRNP genotypes with varying degrees of susceptibility to scrapie and in different brain regions. This study found that PrPC alpha cleavage increased during development from the new born lamb to the adult sheep, which may have consequences for the susceptibility differences related to age. There are also variations in the amount of alpha cleavage between brain regions such as cortex and medulla that may influence scrapie strain targeting. Overall the amount of the C1 fragment in the different brain areas varied as much as 10x (range 5% to 60%). There was a significant difference in the ratio of C1 to the other PrPC forms between two PRNP genotype groups carrying the VRQ and ARQ allele but there was no correlation between C1 level and scrapie susceptibility or scrapie incubation period in our scrapie models. Alpha cleavage of PrPC also occurs in various transgenic mouse models expressing different ruminant PrP sequences. In PrPC over-expressing transgenic mouse models a higher ratio of C1 was observed, this may suggest a link between PrPC expression levels and alpha cleavage. Transgenic mice are therefore important models to further investigate the link between PrPC biology and scrapie disease phenotype. In conclusion, this thesis has shown for the first time that certain ovine PRNP alleles can influence alpha cleavage of the PrPC protein; however it appears not to be a significant indicator of TSE disease susceptibility in sheep.

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