Roles of PIKfyve in the Retina: A Zebrafish Study

Abstract

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.