Modulation of PrPC production and proteolysis for the attenuation of prion disease

  • Author / Creator
    Arce, Luis A
  • Neurodegenerative diseases caused by prions afflict both humans and animals, and result from conformational conversion of the cellular prion protein, PrPC, coded for by the PRNP gene, to an isoform called PrPSc. The infectious agent of PrPSc assembles into aggregate structures and can continue the conversion of PrPC substrate into the disease-causing form. Aggregation of PrPSc causes neurodegeneration and neuronal death. However, to date effective therapeutics are lacking. We set out to attenuate prion disease by modulating expression and processing of PrPC with the rationale that if the substrate for PrPSc is either absent, or is in a non-convertible form, then prion disease progress would be impeded.

    In a first approach we utilised CRISPR/Cas9 technology to knock out PRNP gene expression through a “gene drive” strategy. Gene drives allow for CRISPR/Cas9-mediated edits to be passed on to offspring in an enhanced manner, essentially changing edited genes from a heterozygous state to a homozygous state in all progeny. We initially designed guide RNAs (gRNAs) to target the coding region of PRNP so that Cas9-induced edits would preclude the production of PrPC and generated expression plasmids encoding these designed gRNAs and Cas9 endonuclease. We used the T7E1 mismatch assay but altogether were not able to detect editing events in the selected cell line. We infer that the complexity of targeting, low transfection efficiencies, and restricted sensitivity of editing assessment would have to be overcome for the development of a gene drive for animal prion diseases.
    A second approach to attenuate prion disease is modify PrPC proteolysis. Following posttranslational modification PrPC can remain in full-length form (FL) or can undergo α-cleavage or β-cleavage, generating the C1 or C2 fragments, respectively. Unlike FL and C2 PrP, C1 fragment cannot be converted to PrPSc so increasing α-cleavage or decreasing β-cleavage, could slow disease. We therefore performed a compound library screen to identify PrPC proteolysis agonists/antagonists. Using RK13 cells expressing PrPC we screened a Tocriscreen plus mini library and measured PrPC fragment levels; we initially identified a total of nine compounds that modulated PrPC cleavage, seven of which increased C1 fragmentation and two that decreased C2 fragmentation. However, upon retesting to derive dose-response curves, variability in fragmentation level confounded successful identification of a potential therapeutic modulator of PrPC cleavage.
    Lastly, a third approach used a candidate driven strategy to identify putative α-cleavage proteases (α-PrPases). Based upon the performance of Camostat mesylate, a general (non-class specific) serine protease inhibitor and gene expression profiles we identified six type II membrane proteases as candidates. Transfecting protease expression plasmids in the presence of PrPC substrate, TMPRSS1 (Hepsin) and TMPRSS2 were notable in lowering the amount of both FL and C1 PrP. These two proteases exerted similar effects on other GPI anchored proteins such as Shadoo and Doppel (members of the PrP superfamily) and Thy-1; these data suggest a pathway affecting biogenesis of GPI-anchored proteins that warrants further exploration.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.