Prion diseases are fatal, transmissible neurodegenerative diseases which are associated with the conversion of host encoded prion protein, PrPC, into an aggregated, proteinase-resistant isoform, termed PrPSc. Coding polymorphisms within Prnp, the gene encoding PrPC, are known to affect disease incubation times and susceptibility in human, mouse, and sheep and the most prominent example, codon 129 polymorphism in humans, has major disease modifying effects. However, significant differences in incubation times for scrapie in mice with the same Prnp genotype indicate a major role of PrP-independent genetic factors. To identify these factors, prion-resistant revertants were isolated from a highly prion-susceptible cell clone and the respective transcriptomes were analysed. Remarkably, incubation of revertants with the differentiation agent retinoic acid led to a forty-fold increase in prion propagation rates and downregulation of previously identified differentially expressed genes. This approach led to identification of a gene signature of eighteen genes associated with susceptibility to prion propagation and regulated by differentiation. The experimental validation of the relationship between gene expression and prion propagation by transcriptional silencing confirmed the role of nine genes, expressed in revertants. Several susceptibility genes encode proteins that are involved in the regulation of the extracellular matrix (ECM), a site where disease associated PrP (PrPd) is found. Further experimentation revealed novel insights on how expression of these encoded proteins modulate prion replication mechanistically. Inhibition of fibronectin1 binding to integrin α8 by the RGD peptide significantly decreased the activation of matrix metalloproteinase (MMP)-2/9 whilst prion propagation rates increased. Moreover, Papss2 loss-of-function led to undersulphation of heparan sulphate with a concomitant increase in prion propagation. Remarkably, under these conditions, PrPC was deposited at the ECM. The observation that Neural cell adhesion molecule (Ncam) colocalised with PrPd at the basement membrane of infected, but not of uninfected cells led us to scrutinise membrane microdomains in uninfected and chronically infected cells. Co labelling experiments provided evidence for colocalisation of PrPd with tetraspanin-containing microdomains and GM1 domains. The significance of this finding has yet to be determined. In summary, we have identified a gene regulatory network associated with prion propagation at the ECM which is governed by the cellular differentiation state. Work is currently in progress to determine the functional relevance of the identified genes in vivo.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:668479 |
| Date | January 2015 |
| Creators | Marbiah, M. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1469415/ |
Page generated in 0.0016 seconds