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Title: Defining and understanding the conversion, propagation and trafficking of PrPsc in a prion infected cellular system
Author: Rabbanian, S.
ISNI:       0000 0004 2730 2586
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2011
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Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of normal cellular prion protein (PrPC) to an abnormal disease-associated conformer (PrPSc). The aim of this thesis was to investigate the earliest event in prion infection using a novel cell system. Specifically, it aimed to assess the timescale that PrPC is converted to PrPSc following exposure to RML prions and identify the initial cellular site of PrPSc formation and propagation. The cell biology of the initial events of cellular prion infection are poorly understood since newly formed cellular PrPSc is immunologically indistinguishable from infectious prions in the inocula. As a solution to this problem, an epitope-tag was inserted into the sequence of endogenous PrPC to delineate the formation of de novo PrPSc. A PrP-knock down neuroblastoma cell line was reconstituted with mouse 3F4-, FLAG- and MYC-tagged PrPC. Following identification of cells expressing physiological levels of tagged PrPC, prion-susceptibility was determined by exposure to disease-associated prions. Cells expressing 3F4-tagged PrP, the MYC sequence at position 224 and the FLAG sequence at position 22 or 30 contained PrP resistant to formic acid and proteinase K digestion. A mouse bioassay demonstrated that the PrP-224AlaMYC cell line produce bona-fide infectious epitope-tagged PrPSc on exposure to RML prions. Investigation of de novo tagged PrPSc propagation in this novel cell system demonstrated that cellular prion infection is a dynamic process occurring within one minute of prion exposure and that the plasma membrane is the primary site of prion conversion. It was demonstrated that the late endosomes, lysosomes and endosomal recycling compartments do not appear to be key sites of PrP conversion and prion propagation, whilst the plasma membrane and early endocytic compartments are involved in this key process. The work in this thesis provides new insights into the cell biology of the initial stages of prion conversion and propagation and has implications for neurodegenerative diseases where prion-like mechanisms have been proposed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available