Deriziotis, P.; (2009) Cellular mechanisms in prion-mediated neurodegeneration. Doctoral thesis , UCL (University College London).

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Abstract

Prion diseases are fatal neurodegenerative disorders of both humans and other animals. The cause of prion-mediated neurodegeneration by conversion of the normal cellular prion protein (PrPC) to the disease-related isoform (PrPSc) remains unknown. Increasing evidence suggests a role for the ubiquitin proteasome system (UPS) in prion disease. PrPC and PrPSc isoforms have been shown to accumulate in cells after proteasome inhibition, leading to increased cell death. The aim of this thesis was to investigate the role of cellular degradation systems, such as the UPS and autophagy, in prion-mediated cell death. In UPS-mediated degradation poly-ubiquitinated substrates get degraded by the 26S proteasome, which comprises a 20S hydrolytic core and a 19S regulatory particle. Using a variety of biochemical methods, I report that abnormal beta-sheet-rich PrP isoforms inhibit the catalytic activity of the 26S proteasome, by specifically inhibiting its beta1 and beta5 proteolytic subunits. Pre-incubating these PrP isoforms with an antibody raised against aggregation intermediates abrogates the inhibitory effect seen, consistent with an ‘oligomeric’ inhibitory species. Using open-gated yeast 20S proteasome mutants and conserved 19S ATPase C-terminal peptides containing an essential motif for gate-opening, this thesis describes findings supporting an inhibitory effect on proteasomal gating rather than a direct inhibitory effect on the active sites of the 20S proteasome. These C-terminal peptides open the gate in a ‘key in a lock’ fashion by docking into inter-subunit pockets in the alpha-ring of the 20S proteasome. In this system, the inhibitory effect of the beta-sheet-rich PrP isoforms may be due to abnormal PrP competing with the C-terminal peptides for the inter-subunit pockets, thereby preventing gate-opening. Proteins are also degraded by autophagy, a degradation pathway that has not been adequately characterised in prion disease. This thesis investigates potential roles autophagy may play in prion disease. Data presented here suggest that a) prions are cleared by autophagy, b) prion-infected cells have higher numbers of autophagosomes compared to uninfected controls, c) induction of autophagy ameliorates cell death after proteasome inhibition, indicating cross-talk between the two protein-degradation pathways, and d) it is up-regulated in vivo at end-stage prion disease.

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