Zebrafish Targeted Mutagenesis to Unveil Normal Physiological Functions of, and Interactions between, Prion Protein (PrP) and Amyloid Precursor Protein (APP): Relevance to Alzheimer’s disease

• Author / Creator

  Leighton, Patricia LA

• Much of the work on Prion Protein (PrPC) and Amyloid Precursor Protein (APP) biology has focused on the contributions of their misfolded forms or aggregated metabolites to prion diseases and Alzheimer’s disease, respectively. As subversion/partial loss of some normal functions are also likely contributors to these disease states, it is also important to understand the normal functions of these proteins in healthy organisms. Zebrafish are an attractive model organism for uncovering conserved (and hence important) functions of PrPC and APP because their CNS resembles that of mammals, and their genetic tractability can be harnessed to identify protein functional domains (eg. by ‘rescuing’ a phenotype in loss-of-function mutants with modified mRNAs). Here we created loss-of-function mutants of zebrafish homologs of PrPC and APP (prp1 and appa) to identify normal functions of these proteins, using Tal-Effector Nuclease gene targeting. We also bred the perp-/- mutants to our existing prp2-/- mutants to test for redundancy between prp1 and prp2, and created compound prp1-/-;appa-/- mutants to identify functional interactions between PrPC and APP. We did not observe overt phenotypes in any of the single mutants or compound mutants generated, likely due to genetic and/or physiological redundancy. We went on to challenge the prion protein mutants first with acute loss of a second gene and later with a convulsant. We also looked for subtle phenotypes in neural development by examining an accessible neural tissue in zebrafish larvae- the posterior lateral line, and searched for cognitive deficits in adult prp2-/- mutants using behavioural tests. We found that acute loss of appa, achieved using morpholino gene knockdown, in prp1-/- mutants produced an early developmental phenotype. These developmental defects could be partly reversed or ‘rescued’ by delivering either prp1 mRNA or mouse Prnp to one-cell staged zebrafish embryos. These experiments confirmed our previous finding that prp1 and appa interact genetically. We also found that prp1 and prp2 have redundant roles in modulating neural activity (measured indirectly by quantifying c-fos abundance) during exposure to the convulsant, PTZ. Further, both prp1 and prp2 participate in the development of the zebrafish lateral line neuromasts. Finally, using an object recognition test and novel object approach test, we showed that zebrafish lacking the prp2 paralog have age-dependent deficits in object recognition memory and cognitive appraisal. The zebrafish PrPC and APP loss-of-function mutants and assays that we have developed herein will be used to further dissect the molecular mechanisms through which these proteins participate in neural development, neural activity, and ultimately memory and cognition. For example, ‘rescue’ experiments, wherein modified versions of Prnp and APP mRNA are injected into zebrafish embryos, can be used to determine which PrPC and APP protein domains mediate their normal functions. Such information will be useful for the design of Alzheimer’s disease and prion disease therapeutics.

• Subjects / Keywords

  • prion disease
  • targeted mutagenesis
  • neurodegeneration
  • learning and memory
  • Prion protein
  • neural development
  • neural activity
  • Alzheimer's disease
  • Amyloid precursor protein
  • loss-of-function
  • zebrafish

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3GT5FX6R

• 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.