Roles of PIKfyve in the Retina: A Zebrafish Study

• Author / Creator

  Attia, Karen

• Phosphoinositides (PIs) are membrane lipids that function as signaling molecules. PIs
  undergo phosphorylation and dephosphorylation at various positions to produce the seven known
  PI molecules. PIKfyve is a phosphoinositide kinase that produces phosphoinositide-3,5-
  bisphosphate (PI(3,5)P2) and phosphoinositide-5-phosphate (PI5P). PI(3,5)P2 and PI5P have a
  low abundance in the cell and their roles are not fully characterized. Evidence suggests
  involvement in melanosome biogenesis, phagocytosis, endosomal trafficking, lysosomal
  homeostasis, and autophagy. In humans, mutations in PIKFYVE are rare and have been
  associated with Corneal Fleck Dystrophy and congenital cataracts. Dr. Ian MacDonald at the
  University of Alberta identified a patient with a novel heterozygous missense mutation in
  PIKFYVE (c.5492A>G, p.(His1831Arg)). The patient does not present with corneal flecks nor
  congenital cataracts, but rather exhibits a retinal dystrophy phenotype. Thus, phenotypes
  associated with PIKFYVE mutations in humans are heterogeneous and present largely in the eye.
  Little is known about PIKfyve in vivo due to the embryonic lethality of gene knockout in
  common animal models. Here, I sought to characterize the roles of PIKfyve in vivo to elucidate
  disease mechanism in the patient using zebrafish. I used CRISPR/Cas9 mutagenesis to introduce
  loss of function mutations in pikfyve, and pharmacological inhibition to temporarily inhibit
  Pikfyve. Moreover, I experimented with various precise gene editing technologies in zebrafish to
  create a patient mimic mutant line. I found that Pikfyve inhibition/knockdown impaired retinal
  electrical function, increased cell death, and introduced a variety of abnormalities to the retinal
  pigment epithelium. Moreover, I determined that cytosine base editing is efficient in zebrafish
  and established the technique in our lab.

• Subjects / Keywords

  • PIKfyve
  • Gene Editing
  • Zebrafish
  • Retinal Dystrophy
  • Phosphoinositide
  • CRISPR

• Graduation date

  Fall 2023

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-8kvh-2159

• 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.