Halim, Hazim Arief Bin; (2023) The Mode of Prion Infection of Neuronal cells with extracellular PrP amyloids. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Prion diseases, also referred to as transmissible spongiform encephalopathies (TSEs), are fatal and incurable neurodegenerative disorders. Extracellular prion plaques, composed of disease-associated PrP (PrPD), are found across human and mammalian brain tissues, and interact with the extracellular matrix (ECM). However, it is not known whether matrix-bound prions are released upon ECM remodelling, a process involving degradation of the bioscaffold by proteinases. In turn, prions released from the ECM will potentially infect cells within its vicinity. To investigate the implication of ECM remodelling in prion infection, we developed a decellularised ECM (dECM) model derived from prion-infected iS7 cells to mimic infectious extracellular scaffolding in vivo, and introduced reporter cells with perturbed matrix-degrading function. The dECM was generated via osmolysis of infected iS7 cells, and immunopositive against PrP aggregates as well as resident ECM proteins such as Collagen IV and CSPG4. The infectivity of dECM was equivalent to prion infection with 3.3 x 10-5 of 10% (w/v) RML brain homogenate. Mice inoculated with dECM-infected S7 cell lysates succumbed to scrapie sickness at 171 days post inoculation, only 10 days later than an equivalent amount of persistently prion-infected cells. Furthermore, we demonstrated that following 24 hours of contact with infectious dECM, ECM-bound prion aggregates were mobilised and internalised into 40% of reporter N2a-Prnp-/- cells. To investigate the influence of ECM remodelling in prion infection, we treated uninfected S7 cells seeded onto infectious dECM with siRNA complexes designed to target genes associated with matrix degradation. The knockdown of peptidase-encoding genes; Adam19, Adamts4, or Mmp11, led to a decrease of infection by over 30%. We also examined whether disruption of V-ATPase, a proton pump involved in maturing matrix-degrading enzymes, modulates extracellular infection. Interestingly, the gene knockdown of Atp6ap1, which involves in the synthesis of V-ATPase complex, reduced infection by 45%, and the silencing of both Atp6ap1 and Mmp11 blocked infection by 60%. Furthermore, pharmacological inhibition of V-ATPase using bafilomycin A1 resulted in an approximate 80% block in infection. Thus, this body of work demonstrates that the disruption of prion-embedded matrices promotes infection. This shows that the ECM degradation in prion diseases plays a key role in modulating infection, and targeting the bioscaffold-degrading mechanism will abrogate infection stemming from prion-embedded ECM. We also attempted to develop disease-specific antibodies using subtractive immunisation. This advanced immunisation strategy favours the generation of antibodies against poorly immunogenic epitopes. Unfortunately, since Prnp null mice had shown severe complications following immunisations using antigens including PrP peptides and prion-infected exosomes, this project was discontinued.

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