The transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases of the CNS in humans and animals, characterised by long incubation periods and transmissibility within and between different species. The primary aims of this thesis were firstly to analyse the possible role of caspase- independent apoptotic cell death pathways in TSEs, and secondly to develop an in vitro model of neuronal loss in order to assess the relationship between amyloid fibril structure and neurotoxicity.
Caspase- independent pathways were analysed in two well characterised murine scrapie models (ME7 /CV and 87V/VM). Whole or micro -dissected brain areas from terminal stage infected mice were separated into cytosolic and mitochondrial fractions, and their protein contents compared by Western blot to normal brain injected age- matched control animals. Micro -dissection proved to be a much more sensitive technique for the detection of apoptosis -related proteins in vivo. Caspase- independent apoptosis inducing factor (AIF) was found to translocate from the inner mitochondrial membrane space (IMM) to the cytosol in ME7 /CV animals, but not in 87V/VM or in normal brain -injected control cohorts. The release of AIF into the cytosol is therefore specific to infection with the ME7 murine scrapie strain. Furthermore, cytochrome c (cyt c) was released from the IMM in all animals at the terminal stage of disease. However, cyt c was not released from the IMM in younger animals, indicating that dysfunction of the mitochondria is related to age and not to disease.
A neuronal PC 12 cell system was established to model TSE neurodegeneration in vitro. Murine PrP105 -125, homologous to the neurotoxic human PrP106 -126 synthetic peptide, was developed specifically for this research. Mature amyloid fibrils were created containing f3-sheet structures, but different tertiary structures as revealed XV
by electron microscopy, Thioflavin T binding assays and FT -IR techniques. The primary structure of the MoPrP105 -125 peptide is critical for conferring the neurotoxicity, as a scrambled sequence is not toxic to neurons. Furthermore, the research within this thesis shows that the morphology of mature amyloid fibrils does not have a significant effect on toxicity, suggesting that the intermediate soluble protofibrillar structures may prove to be the more toxic species
n conclusion, this research shows that the mechanisms of neuronal loss in two tested TSE models can follow different biochemical pathways, which might explain differences in the selective targeting of these TSE agents to different CNS neuronal populations. These studies also revealed that mitochondrial dysfunction occurred within the lifespan of the animals tested in this research, which may contribute to neuronal death in the ME7 /CV model. In addition, an in vitro model demonstrated that PrP fibril toxicity was principally related to the primary sequence of the fibrils, and not to the secondary or tertiary structures of mature fibrils. These results are all relevant to the future development of potential therapeutic strategies for TSEs, and may also have wider implications for the treatment of other CNS amyloidogenic diseases.