Disrupting the Repeat Domain in Zebrafish Premelanosome Protein (Pmela) to Probe an Evolutionary Puzzle and Model Pigmentary Glaucoma

• Author / Creator

  Hodges, Elizabeth D.

• The premelanosome protein (PMEL) is a functional amyloid that provides a scaffold for
  the even distribution of melanin and provides structure for the organelle that contains them, the
  melanosome. Mutations in this protein cause a plethora of phenotypes that extend beyond
  affecting strictly pigmentation. Dominantly inherited, non-synonymous point mutations in the
  repeat domain of the premelanosome protein (PMEL) cause pigmentary glaucoma in humans.
  To better appreciate PMEL’s biology and molecular complexity, we first positioned
  PMEL within an evolutionary context by comparing species, various mutations, and other related
  genes (GPNMB, TMEM130). We focused our attention on PMEL’s repeat domain, because it is
  the location of many of the human mutations and is a known contributor to the functional
  amyloid structure. We hypothesize that PMEL’s repeat domain is necessary for normal
  pigmentation and ocular anatomy and function. To assess this, lab members mutated the
  repetitive domain in zebrafish PMEL.
  Prior to the collaborative efforts presented in this thesis, existing animal models with
  PMEL mutations were null mutants that exhibit recessive inheritance. Due to poor primary
  sequence conservation (despite functional conservation) in PMEL’s repeat domain, the point
  mutations observed in humans are difficult to model in animals. Our lab generated zebrafish with
  an in-frame deletion within the repeat domain to test the hypothesis that the repeat domain is
  required for melanosome function. We compare this new mutant (with a perturbation restricted
  to the repeat region) to wildtype and a pmela null (or strong hypomorph) mutant.
  This new mutant contrasts the aforementioned pmela null mutant zebrafish in that it is
  predicted to produce (modified) premelanosome protein. Moreover, we observed dominant
  inheritance with a phenotype in heterozygous animals. Both mutations cause larval melanosomal

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  and ocular phenotypes. Neither of the adult zebrafish mutants had ocular phenotypes when
  clinically evaluated with optical coherence tomography (OCT) and rebound tonometry. Use of
  rebound tonometry in zebrafish is described, and numerous baseline measures were taken. This
  provides an easy tool to assess intraocular pressure, a hallmark of glaucoma, in zebrafish models.
  In conclusion, we found that disrupting the repeat region was sufficient to cause
  pigmentary and ocular pathology in zebrafish and, curiously, through mechanisms that did not
  align perfectly with the previously described loss of function mutations. This new repeat domain
  mutant provides a tool to look at the effect of the repeat region on ocular development and in
  vivo fibril structure.

• Subjects / Keywords

  • premelanosome protein
  • glaucoma
  • zebrafish

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-tgtq-be38

• License

  This thesis is made available by the University of Alberta Library 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.